Compositions and methods for treatment in broad-spectrum, undifferentiated or mixed clinical applications

ABSTRACT

The disclosure provides improved compositions and methods for passive immunization. In embodiments, a composition comprising a synergistic combination of specific polyclonal antibodies in a carrier matrix is provided. The disclosure provides effective, economical compositions and methods for the treatment of diarrhea and enteric infections in broad-spectrum, undifferentiated, or mixed clinical applications.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/616,724, filed Jun. 7, 2017, which is a continuation of U.S.application Ser. No. 13/302,836, filed Nov. 22, 2011, now U.S. Pat. No.9,701,735, issued Jul. 11, 2017, which claims the benefit of U.S.Provisional Application No. 61/416,667, filed Nov. 23, 2010, to TimothyW. Starzl, of Boulder, Colo., entitled “Compositions and Methods forTreatment in Broad-Spectrum, Undifferentiated or Mixed ClinicalApplications”, the entire contents of each of which is incorporatedherein by reference.

FIELD OF THE DISCLOSURE

The disclosure provides compositions and methods for passiveimmunization. In embodiments, compositions comprising a synergisticcombination of specific polyclonal antibodies with a carrier matrix areprovided. The disclosure provides effective and economical compositionsand methods for the treatment of pathogenic infections inbroad-spectrum, undifferentiated, or mixed clinical applications. In oneembodiment, compositions and methods for the treatment of diarrhea andenteric infections are provided.

BACKGROUND OF THE DISCLOSURE

Antibodies, immunoglobulins, and other biological immune factors(referred to here collectively as antibodies), both natural and theirsynthetic analogues, are known therapeutic agents in humans and animals.Antibodies operate by binding (via non-covalent forces) between theantigen combining site on the antibody and a portion of the antigencalled the antigenic determinant or epitope. Antibodies are capable ofhigh degrees of specificity. For example, the field of monoclonalantibodies has developed largely under the impetus of producing evermore specific and precise binding characteristics. However, this highspecificity can lead to excessively limited binding attributes, whereagents or antigens that are functionally identical do not reactidentically with the immunoreagent or immunotherapeutic.Cross-reactivity on the other hand, usually considered an error orfailure to achieve binding specificity., is the reaction between anantigen and an antibody that was generated against a similar butdifferent antigen. Controlled cross-reactivity may constructively beused to broaden the binding range of the antibody.

Colostrum has evolved naturally in mammals specifically to deliver itscomponents to neonates to and through the gastrointestinal tract in avery concentrated low-volume form. Colostrum is known to containantibodies such as IgA, IgG, and IgM. Other components of colostruminclude lactoferrin, lysozyme, lactoperoxidase, complement, andproline-rich polypeptides (PRP). A number of cytokines (small messengerpeptides that control the functioning of the immune system) are found incolostrum as well, including interleukins, tumor necrosis factor,chemokines, and others. Colostrum also contains a number of growthfactors, such as insulin-like growth factors I, and II, transforminggrowth factors alpha, beta 1 and beta 2, fibroblast growth factors,epidermal growth factor, granulocyte-macrophage stimulating growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, and colony-stimulating factor-I.

The antibodies and cofactors in colostrum can, through breast feedingprovide a passive immunity to the recipient. Normally antibodies andcofactors are passed to the neonate from the mother and provide thefirst protection against pathogens. Growth factors also stimulate thedevelopment and repair of the gut.

One condition that could be addressed by using passive immunity isdiarrhea. Diarrhea is caused mainly by the ingestion of pathogens.According to the World Health Organization (WHO), eighty-eight percentof cases diarrhea worldwide are attributable to unsafe water, inadequatesanitation or insufficient hygiene. These cases result in about 1.5million deaths each year, most being the deaths of children.(Pruss-Urstun et al., Safer water, better health: costs, benefits andsustainability of interventions to protect and promote world health.World Health Organization, Geneva, 2008. ISBN 978 92 4 1596435).

Of particular global concern are the instances of infectious diarrhea inthe developing world, which are a cause of tremendous ongoing morbidityand mortality, particularly in the pediatric population. For example,India has one of the highest infant mortality rates in the worldaccording to a 2009 United Nations Human Development report. Forexample, Save the Children, a global non-profit, reports that one childdies every 15 seconds in India, and 90% of these deaths are due topreventable diseases, such as diarrhea. Rotavirus and measles vaccines,handwashing with soap, improved drinking water supply and community-widesanitation are recommended by WHO for the prevention of diarrhea;however, these measures are not effective to treat the disease.

Standard treatment protocol in much of the world for pediatric diarrheaincludes a concomitant administration of antibiotics and oralrehydrative therapy. For many reasons, antibiotics are a prescriptiondrug. Antibiotics are not effective in the treatment of viral infection.For example, rotavirus is estimated to cause about 40 percent of allhospital admissions due to diarrhea among children under five years ofage worldwide. (Weekly Epidemiological Record, vol. 83, no. 47, 21 Nov.2008). The inappropriate use of antibiotics can promote resistantstrains of bacteria. Conversely, the infection may be caused by aresistant strain of bacteria. Even under the best of circumstances, useof an appropriate antibiotic may take several days to reduce theseverity of the symptoms of diarrhea.

Another disadvantage of antibiotics is that administration can inducethe destruction of both pathogenic and benign bacteria found in the GItract which can further result in release of endotoxiclipopolysaccharides. (Holzheimer, The significance of endotoxin releasein experimental and clinical sepsis in surgical patients—evidence forantibiotic-induced endotoxin release? Infection. 1998 March-April;26(2):77-84). These endotoxins have a host of adverse systemic effectsincluding fever, changes in white blood cell counts, disseminatedintravascular coagulation, hypotension, shock and death, malabsorption;in fact, the direct injection of fairly small doses of endotoxin resultsin death in most mammals. Todar K. Bacterial Endotoxin. Textbook ofBacteriology. 2008. textbookofbacteriology.net.

According to WHO, oral rehydration therapy and zinc with continuedfeeding, including breastfeeding, is recommended for treatment ofchildhood diarrhea. Zinc syrup or zinc-fortified oral rehydrationsolution (ORS, 40 mg/L) is typically employed at a dose of about 15 to30 mg per day. Zinc is inexpensive, but has modest efficacy. Zinc syrupresults in only about a 25 percent reduction in duration of acutediarrhea, and a 40 percent reduction in treatment failure or death.(Bhutta et al. Therapeutic effects of oral zinc in acute and persistentdiarrhea in children in developing countries: pooled analysis ofrandomized controlled trials. The American Journal of ClinicalNutrition. 2000; 72(6):1516-22). One study evaluated the efficacy andsafety of a zinc-fortified (40 mg/L) ORS among 1,219 children with acutediarrhea. Clinical outcomes among the zinc-fortified ORS group weremodestly improved, compared with those for the control group, whoreceived standard ORS only. In that study, the total number of stoolswas lower among the zinc-ORS group compared with the total number forthe control group. No substantial effect on duration of diarrhea or riskfor prolonged diarrhea was noted. (Bahl R, Bhandari N, Saksena M, et al.Efficacy of zinc-fortified oral rehydration solution in 6- to35-month-old children with acute diarrhea. J Pediatr 2002; 141:677-82).

It is known that antibiotics are ineffective to treat a viral infection,such as a rotavirus infection. Other interventions have limitedeffectiveness. Additionally, appropriate diagnostic tools to distinguishthe cause of diarrhea are not always readily available or affordable.

Clearly a rapid, effective and economical alternative for the treatmentof undifferentiated diarrhea is desirable. There remains a need foreffective, economical compositions and methods for treatment of diarrheaand enteric infections in broad-spectrum, undifferentiated, or mixedclinical applications.

SUMMARY OF THE DISCLOSURE

The disclosure provides compositions and methods of passive immunizationwherein a specific binding molecule, such as a specific immunoglobulin,is combined with a carrier matrix to provide a composition for oral ormucosal administration for management of microorganisms; includingtreatment or prophylaxis of a pathogenic infection or undesirablestrain. In embodiments, the compositions are administered to anon-neonatal subject.

In one embodiment, the disclosure provides a composition foradministration to a non-neonate human for the management ofmicroorganisms, the composition comprising at least one specific bindingmolecule, or fragment thereof, derived from the adaptive immune systemof an animal, wherein the specific binding molecule is selected from animmunoglobulin, antibody, peptide, variable lymphocyte receptor,transfer factor, or a mixture thereof; and a carrier matrix comprisingtwo or more components of the innate immune system of a non-humanmammal, wherein the matrix can be selected from, or derived from thecomponents of, colostrum, milk, serum, plasma, saliva, lymph fluid,mucous, or lachrymal fluid; wherein the matrix and the specific bindingmolecule are derived from different species.

In a preferred embodiment, the carrier matrix comprises bovinecolostrum. In another embodiment, the matrix comprises the components ofthe innate immune system that are selected from lysozyme, phospholipase,defensins, opsonins, components of the complement system, beta-lysin,protein-rich peptides (PRP), (PRPs), lactoferrin, transferrin,cytokines, interleukins, chemokines, interferons, TNF-alpha,fibronectin, leukocytes, white blood cells, phagocytes, macrophages,monocytes, neutrophils, polymorphonuclear cells, dendritic cells, mastcells, eosinophils, basophils, natural killer (NK) cells, lymphokineactivated killer (LAK) cells, defensins, elastase, cathepsin G,myeloperoxidase, and NADPH oxidase.

In various embodiments, the composition includes a pharmaceuticallyacceptable carrier. In other embodiments, the composition includes afood grade carrier. In embodiments, the compositions can be administeredvia oral delivery, nasal delivery, ocular delivery or combinationsthereof.

In other embodiments, the composition does not include an exogenouslyadded polymer, copolymer, liposome, hydrogel or fibrin. In otherembodiments, the composition does not include microspheres ormicrocapsules. In yet a further embodiment, the composition does notinclude an exogenously added antigen.

In a further embodiment, the specific binding molecules specificallybind to a pathogen, a pathogen related toxin, a pathogen relatedadhesion element, undesirable strain, or a combination thereof. In oneaspect, the pathogen comprises a human or veterinary, enteric orgastrointestinal, pathogen causing gastroenteritis.

In various aspects, the pathogen or undesirable strain is selected fromthe group consisting of: Campylobacter jejuni, Salmonella, Salmonellaenterica serovar Typhi, Shigella dystenteriae, Plesiomonas shigelloides,Escherichia coli [including (EPEC) enteropathogenic E. coli, (ETEC)enterotoxigenic E. coli, (EaggEC) enteroaggregative E. coli, (EIEC)enteroinvasive E. coli, and (EHEC) haemorrhagic E. coli], Clostridiumdifficile, Yersinia enterocolitica, Vibrio cholerae O1, Vibrio O139,Non-O1 Vibrios, Vibrio parahaemolyticus, Aeromonas hydrophila,Clostridium perfringens, Clostridium difficile, enterohepaticHelicobacter (including Helicobacter pylori), Staphylococcus aureus,Klebsiella, rotavirus, coronavirus, norovirus, calicivirus, entericadenovirus, cytomegalovirus, astrovirus, S. pneumoniae, H. influenzae,Neisseria gonorrhoeae, herpes zoster virus, Fusarium spp., andAcanthamoeba spp.

In a specific aspect, the pathogen related toxin comprises an endotoxinor exotoxin.

In another specific aspect, the pathogen related adhesion elementcomprises adhesins, cadherins, cilia, fimbrillae, a viral adhesionstructure, or a combination thereof.

In various embodiments, the composition is administered orally in anamount effective for the treatment or prevention of undifferentiateddiarrhea, traveler's diarrhea, rotavirus diarrhea, toxin-mediateddiarrhea, cholera, C. difficile infection, dysentery, typhoid fever,peptic ulcers, or for gastrointestinal flora management. In anotheraspect, an effective amount of the composition confers passive immunityto a subject.

In another embodiment, the disclosure provides a method for preparingthe composition of the disclosure by the steps of: (a) obtaining from ananimal at least one specific binding molecule or fragment thereof thatbinds to a specific antigen, wherein the specific binding molecule isselected from an immunoglobulin, antibody, peptide, variable lymphocytereceptor, transfer factor, and a mixture thereof; (b) obtaining at leastone carrier matrix, comprising at least two components obtained from anonhuman animal selected from the group consisting of enzymes,lactoferrin, transferrin, nonspecific immunoglobulins, cytokines, whiteblood cells, complement components, interferons, and fibronectin; (c)preparing a solid form of the carrier matrix and of the specific bindingmolecule or fragment thereof; and (d) mixing the solid form of thecarrier matrix with the solid form of the specific binding molecule orfragment thereof.

In another embodiment, the present invention provides a method forpreparing an immunity conferring composition. The method includes (a)obtaining at least one exogenously sourced specifically targeted immunefactor; (b) preparing a powderized form of the at least one exogenouslysourced specifically targeted immune factor; (c) obtaining at least oneexogenously sourced carrier matrix, optionally mixed the exogenouslysourced carrier matrix with a mixture of agents to support and interactwith the exogenously sourced specifically targeted immune factor; (d)preparing a powderized form of the at least one exogenously sourcedcarrier matrix; and (e) mixing the powderized form of step (b) with thepowderized form of step (d), thereby obtaining the passive immunityconferring composition. In one aspect, the passive immunity conferringcomposition includes a dose controlled formulation. In various aspects,the passive immunity conferring composition includes a pharmaceuticallyacceptable carrier. In various aspects, the passive immunity conferringcomposition does not include a polymer, copolymer, liposome, hydrogel,or fibrin. In various aspects, the passive immunity conferringcomposition does not include microspheres or microcapsules. In variousaspects, the passive immunity conferring composition does not include animmunogen or antigen.

The present invention includes at least one of the followingdistinguishing attributes: (a) it enables customized design of thematrix, specific factors, and the activating events for specified ortargeted diseases; (b) it enables dose controlled formulation of avariety of mixtures of components, which may be tuned or adjusted foreffect; (c) it enables dose controlled formulation that providesspecified components in excess of normal physiological levels that canbe achieved in natural systems; (d) it uses complex multi-componentmulti-pathway interactions to create a systems effect that emulates anative immune system response; (e) it enables creation of apreconditioned or potentiated passive immune response that can beadministered in its potentiated state, and subsequently activated by thepresence of the target pathogens, toxins, disease state, or syndrome;(f) it enables the creation of formulations that have a definedspecificity or broad-spectrum effect, to match the needs of the specifictarget disease state or syndrome, or of the practice environment withinwhich the product is to be used; and (g) it enables the creation offormulations that can be targeted for prophylaxis as well as fortherapeutic intervention.

In another aspect, by adjusting the amounts of the specific bindingmolecules, such as polyclonal antibodies, in the composition a dosecontrolled formulation can be prepared.

In a preferred embodiment, the at least one specific binding moleculecomprises IgY derived from immunized chickens In other specific aspects,the IgY comprises a pool of IgY specific for at least enterotoxigenic E.coli spp., E. coli K99 pili adherence factor, Clostridium perfringenstoxoid, Salmonella typhimurium, rotavirus, and coronavirus.

In another embodiment, the composition is topically administered to amucosal membrane.

In another embodiment, the pathogen comprises a pathogen causingvaginitis. In various aspects, the pathogen is selected from the groupconsisting of: Gardnerella spp., Neisseria gonorrhoeae, Chlamydiaceaetrachomatis, Mycoplasma spp., Campylobacter jejuni, Trichomonasvaginalis, herpes virus type 1, herpes virus type 2, Candida albicans,Candida glabrata, Candida tropicalis, Candida parapsilosis and Candidakrusei.

In another embodiment, the pathogen is Group A Streptococcus bacteria.

In another embodiment, the pathogen comprises a pathogen causingconjunctivitis selected from the group consisting of S. aureus, S.pneumoniae, H. influenzae, Neisseria gonorrhoeae, Chlamydia trachomatis,adenovirus, herpes simplex, herpes zoster virus, enteroviruses, Fusariumspp, Candida spp. and Acanthamoeba spp.

In another embodiment, the compositions of the disclosure are useful asnutritional compositions for administering to a subject in need thereofwho is afflicted with a disease that creates special dietary needs suchas pediatric diarrhea, Crohn's disease, and ulcerative colitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows average daily stool frequency over a five day period fortwo field trial test groups compared to negative control for thecomposition of Example 1A. In Trial 1 (n=29) and Trial 2 (n=31), thecomposition Example 1A is administered once daily for three days withantibiotic and oral rehydration salts (ORS). In the Negative Control(n=28), only antibiotic and ORS are administered without a compositionof the disclosure.

FIG. 2 shows average daily stool consistency on a 1-5 scale (1=normaland 5=liquid) over the same five day period for the same three groupsfrom FIG. 1.

FIG. 3 shows average physician assessed wellbeing on a 1-5 scale(1=normal and 5=severely ill) over the same five day period for the samethree groups from FIG. 1.

FIG. 4 shows average daily stool frequency over a five day period forthree field study (trial) test groups. Trials 1 and 2, and the negativecontrol, are as described for FIG. 1. In Trial 3 (n=140), patients wereadministered the composition of Example 1B with antibiotic and ORS.

FIG. 5 shows average daily stool consistency on a 1-5 scale (1=normaland 5=liquid) over the same five day period for the same four groupsfrom FIG. 4.

FIG. 6 shows average physician assessed wellbeing on a 1-5 scale(1=normal and 5=severely ill) over the same five day period for the samefour groups from FIG. 1.

FIG. 7 shows average daily stool frequency over a five day period forTrials 1 and 2, negative control, Trial 3 is broken into 6 subgroups(ES204A): 2 g spray dried egg with 4 g colostrum administered for 3days; (ES204B): 2 g spray dried egg with 4 g colostrum administered for2 days; (MT204A) 2 g thermal dried egg with 4 g colostrum for 3 days;(MT304A) 3 g thermal dried egg with 4 g colostrum for 3 days; (MS204A) 2g spray dried egg with 4 g colostrum for 3 days; (MS304A) 3 g spraydried egg with 4 g colostrum for 3 days.

FIG. 8 shows average daily stool consistency on a 1-5 scale (1=normaland 5=liquid) over the same five day period for the same groups fromFIG. 7.

FIG. 9 shows average physician assessed well-being on a 1-5 scale(1=normal and 5=severely ill) over the same five day period for the samegroups from FIG. 7.

DETAILED DESCRIPTION Definitions

The terms “prevention”, “prevent”, “preventing”, “prophylaxis” and asused herein refer to a course of action (such as administering acompound or pharmaceutical composition of the present disclosure)initiated prior to the onset of a clinical manifestation of a diseasestate or condition so as to prevent or reduce such clinicalmanifestation of the disease state or condition. Such preventing andsuppressing need not be absolute to be useful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a clinical manifestation of adisease state or condition so as to eliminate or reduce such clinicalmanifestation of the disease state or condition. Such treating need notbe absolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method, compound or pharmaceutical composition of thedisclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method, compound or pharmaceuticalcomposition of the disclosure.

The term “individual”, “subject” or “patient” as used herein refers toany animal, including birds or mammals, such as mice, Norway rats,cotton rats, gerbils, cavies, hamsters, other rodents, rabbits, dogs,cats, swine, cattle, sheep, goat, horses, or primates, and humans. Theterm may specify male or female or both, or exclude male or female. Inone aspect, the patient is an adult human. In another aspect, thepatient is a non-neonate human infant. In another aspect, the patient isa human toddler, child, or adolescent.

The term “neonate”, or newborn, refers to an infant in the first 28 daysafter birth. The term “non-neonate” refers to an animal older than 28days.

The term “effective amount” as used herein refers to an amount of anagent, either alone or as a part of a pharmaceutical composition, thatis capable of having any detectable, positive effect on any symptom,aspect, or characteristics of a disease state or condition. Such effectneed not be absolute to be beneficial.

The term “including” as used herein is non-limiting in scope, such thatadditional elements are contemplated as being possible in addition tothose listed; this term may be read in any instance as “including, butnot limited to.”

The term “immunize”, “actively immunize”, “actively immunizing”, and“active immunization” means to purposefully immunize a subject byexposing a subject to an antigen, for example, an antigen derived from amicroorganism, such as but not limited to, a virus or a bacteria; suchexposure may be carried out by exposing the subject to an intactorganism, an attenuated organism, a portion of the organism, one or moreantigens present on the organism or a combination of the foregoing.

The term “passively immunize”, “passively immunizing”, and “passiveimmunization” means to provide antibodies against an antigen, forexample, an antigen derived from a microorganism, such as but notlimited to, a virus or a bacteria, to a subject without necessarilyeliciting an immune response to the organism in the subject. Passiveimmunization provides immediate protection but the subject does notdevelop memory cells as a result.

The term “passive immunity” as used herein refers to artificiallyacquired immunity achieved by the transfer of antibodies to the subject.The terms “egg” or “egg product” each mean an avian sourced whole shellegg (conventional, immunized or otherwise) or any product or fractionderived therefrom.

The terms “immune egg” or “immune egg product” each mean whole egg orany product or fraction derived therefrom, obtained from an eggproducing animal maintained in a immunized state.

The term “antigen” refers to an entity or fragment thereof which caninduce an immune response in an organism, particularly an animal. Theterm includes immunogens and regions thereof responsible forantigenicity or antigenic determinants.

The term “polyclonal antibody” refers to antibodies that areheterogeneous populations of antibody molecules derived from the sera ofanimals immunized with an antigen or an antigenic functional derivativethereof. For the production of polyclonal antibodies, various hostanimals may be immunized by injection with the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species.

The term “monoclonal antibody” is well recognized in the art and refersto an antibody that is mass produced in the laboratory from a singleclone and that recognizes only one antigen. Monoclonal antibodies aretypically made by fusing a normally short-lived, antibody-producing Bcell to a fast-growing cell, such as a cancer cell (sometimes referredto as an “immortal” cell). The resulting hybrid cell, or hybridoma,multiplies rapidly, creating a clone that produces large quantities ofthe antibody. “Monoclonal antibodies” are substantially homogenouspopulations of antibodies directed to a particular antigen or epitope.They may be obtained by any technique which provides for the productionof antibody molecules by continuous cell lines in culture. Monoclonalantibodies may be obtained by methods known to those skilled in the art.See, for example, Kohler, et al., Nature 256:495-497, 1975, and U.S.Pat. No. 4,376,110.

The term “crystalline” refers to an antibody, such as a monoclonalantibody that has been purified by crystallization, such as by batchcrystallization. Crystalline antibodies can be used in order to generatea small volume, highly concentrated forms. (Yang et al., 2003,Crystallline antibodies for subcutaneous delivery. PNAS100(12):6934-6939).

The term “undifferentiated diarrhea” means that the causative agent ofthe diarrhea is undiagnosed.

The term “antibody fragment” encompasses any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments, “Fv” fragments, consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“scFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion. Antibody fragments include a portion of an antibody such asF(ab′)2, F(ab)2, Fab′, Fab, Fv, sFv and the like. Regardless ofstructure, an antibody fragment binds with the same antigen that isrecognized by the intact antibody.

The term “transfer factor” refers to an immune molecule of approximately5000 Daltons, made up of amino acids, that cause antigen-specificcell-mediated immunity, primarily delayed hypersensitivity and theproduction of lymphokines, as well as binding to the antigensthemselves. (Kirkpatrick 1993, Structural nature and functions oftransfer factors. Ann. N.Y. Acad. Sci. 685:362-368.)

The term “variable lymphocyte receptors” refers to lymphocyte-derivedmolecules discovered in jawless vertebrates such as the lamprey andhagfish. These animals possess a large array of variable lymphocytereceptors that are produced from only a small number of genes and thatbind to pathogenic antigens in a similar way to antibodies, and with thesame degree of specificity. (Alder et al., 2005, Diversity and functionof adaptive immune receptors in a jawless vertebrate. Science,310(5756):1970-1973).

The term “cell receptor” refers to the ligand binding moiety of theB-cell receptor; a membrane bound immunoglobulin molecule of one isotype(e.g., IgD, IgM, IgE). With the exception of the presence of an integralmembrane domain, these are identical to their secreted forms.

The term “specific binding” in the context of the characteristics ofspecific binding molecules, also known as specific targeted immunefactors, such as an antibody, antibody fragment, variable lymphocytereceptor, or transfer factor, refers to the ability to preferentiallybind to a particular antigen that is present in a homogeneous mixture ofdifferent antigens. In certain embodiments, a specific bindinginteraction will discriminate between desirable and undesirable antigens(e.g., “target” and “non-target” antigens) in a sample, in someembodiments more than about 10 to 100-fold or more (e.g., more thanabout 1000- or 10,000-fold). In some embodiments, the specific bindingmolecule may specifically bind to an epitope shared among differentspecies or strains of a microorganism as compared to non-sharedepitopes. In certain embodiments, the affinity between an antibody andantigen when they are specifically bound in an antibody-antigen complexis characterized by a K_(D) (dissociation constant) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸M, less than 10⁻⁹M, less than 10⁻¹⁰ M,less than 10⁻¹¹ M, or less than about 10⁻¹²M or less.

The term “innate immune system”, or non-specific immune system, refersto the cells, molecular components and mechanisms that defend the hostfrom infection by other organisms in a non-specific manner. The cellsand molecular components of the innate immune system recognize andrespond to pathogens in a generic way, but unlike the adaptive immunesystem, it does not confer long-lasting or protective immunity to thesubject. Innate immune systems provide immediate defense againstinfection. Vertebrates possess a second layer of protection, theadaptive immune system, which is activated by the innate response.

The term “adaptive immune system” refers to highly specialized, systemiccells and processes that recognize and respond to an antigen, forexample, to eliminate, neutralize or prevent pathogenic growth. Thesystem is highly adaptable due to somatic hypermutation (a process ofaccelerated somatic mutation) and V(D)J recombination (an irreversiblegenetic recombination of antigen receptor gene segments). Adaptiveimmunity is also referred to as acquired immunity and creates animmunological memory. An adaptive immune response is pathogen andantigen specific and there is a lag time between exposure and maximalresponse. An adaptive immune response is based on the principle ofclonal recognition, such that upon first exposure to an antigen, primedlymphocytes either differentiate into immune effector cells or form anexpanded pool of memory cells that respond to secondary exposure to thesame antigen by mounting an amplified and more rapid response.

The term “animal” refers to the animal kingdom definition.

All pronouns are intended to be given their broadest meaning. Unlessstated otherwise, female pronouns encompass the male, male pronounsencompass the female, singular pronouns encompass the plural, and pluralpronouns encompass the singular.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

Modes of the Disclosure

The disclosure provides compositions and methods useful in themanagement of undesirable strains or pathogenic microorganisms.

One embodiment of the present invention is based on a method to create atargeted antibody-based formulation embedded or subsumed within acarrier matrix, where the antibodies use a controlled form ofcross-reactivity to multiple clusters of related target antigens, andwhere the carrier matrix contains support and cofactors that enhance theeffect of the antibodies. The utility of such antibody/matrixformulations may include providing broad-spectrum therapeuticinterventions under conditions where the class of causative agent, butnot the precise or specific causative agent, is known or suspected orunder circumstances where multiple (mixed) causative agents are active.

A novel approach to the use of antibodies in this manner has beendeveloped, that takes advantage of both the specificity andcross-reactive attributes of antibodies, and then further utilizes thecomponents within the carrier matrix to generate a multi-component insitu immune response. In this embodiment, antibodies are designed tobind to all of several closely related epitopes that represent astructurally related cluster of antigens. These antigens may differmarkedly in other respects, and may originate from diverse sources,organisms, or species.

One embodiment of the invention involves the method of specific bindingmolecules (immune factors, for example antibodies), within the carriermatrix, where the specific binding molecules have specificity to a classof related antigens, and are specifically cross-reactive to differentinstances of members of that class. There exists a degree of structuralsimilarity in related clusters of target antigens, without regard to theorganism or pathogen that is the source of the antigen. Similarity instructure can result in a phenomenon known as “cross-reactivity” (thesteric binding of a reactive molecule to an antigen other than theantigen intended). Cross-reactivity is often unintentional, and in mostcases is considered a source of error and non-specificity. However, inthis embodiment the extent and degree of cross-reactivity is controlledby various means to limit and channel its expression so as to providedesired characteristics.

This treatment confers passive immunity to patients. The nature of thetreatment makes the associated risk factors comparable to that of eatingfood from the source where the antibodies were harvested (e.g., riskfactors would be similar to that of eating an egg and a glass of milk).This is an effective treatment with less toxicity than the currentlyavailable alternative interventions.

The present invention is based on the seminal discovery that the use ofan exogenously sourced (containing components obtained from an animaldifferent from the animal to be treated) carrier matrix in conjunctionwith exogenously sourced (obtained or corresponding to immune factorsobtained from an animal different form the animal to be treated)specifically targeted immune factors can be used to transport andintroduce an effective multi-parameter immunity to a subject in needthereof.

In one aspect, the disclosure provides a composition comprising: a) anon-neonate human effective amount of at least one specific bindingmolecule, or fragment thereof obtained from an animal and thatspecifically binds to an antigen, wherein the specific binding moleculeis selected from an immunoglobulin, antibody, peptide, variablelymphocyte receptor, transfer factor, and a mixture thereof; and, b) acarrier matrix comprising at least two components obtained from anonhuman animal selected from the group consisting of enzymes,lactoferrin, trnasferrin, nonspecific immunoglobulins, cytokines, whiteblood cells, complement components, interferons, and fibronectin,wherein the at least one specific binding molecule and the at least twocomponents of the carrier matrix are obtained from different animals.

In another aspect, the disclosure provides a method for preparing thecomposition comprising: (a) obtaining at least one specific bindingmolecule or fragment thereof from an animal that binds to a specificantigen, wherein the specific binding molecule is selected from animmunoglobulin, antibody, peptide, variable lymphocyte receptor,transfer factor, and a mixture thereof; (b) obtaining at least onecarrier matrix, comprising at least two components obtained from anonhuman animal selected from the group consisting of enzymes,lactoferrin, trnasferrin, nonspecific immunoglobulins, cytokines, whiteblood cells, complement components, interferons, and fibronectin; (c)preparing a solid form of the carrier matrix and of the specific bindingmolecule or fragment thereof; and (d) mixing the solid form of thecarrier matrix with the solid form of the specific binding molecule orfragment thereof.

In yet another aspect, the compositions of the disclosure are useful inthe treatment or prevention of microbial infections. In embodiments,microbial infections include those caused by Campylobacter jejuni,Salmonella, Salmonella enterica serovar Typhi, Shigella dystenteriae,Plesiomonas shigelloides, Escherichia coli, enteropathogenic E. coli,enterotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E.coli, haemorrhagic E. coli, Clostridium difficile, Yersiniaenterocolitica, Vibrio cholerae O1, Vibrio O139, Non-O1 Vibrios, Vibrioparahaemolyticus, Aeromonas hydrophila, Clostridium perfringens,enterohepatic Helicobacter, Helicobacter pylori, Staphylococcus aureus,Klebsiella, rotavirus, coronavirus, norovirus, calicivirus, entericadenovirus, cytomegalovirus, and astrovirus. In embodiments, thecompositions are useful to treat or prevent conditions such asundifferentiated diarrhea, traveler's diarrhea, rotavirus diarrhea,toxin-mediated diarrhea, cholera, C. difficile infection, dysentery,typhoid fever, peptic ulcers, vaginitis, or for gastrointestinal floramanagement.

In a specific embodiment, the compositions and methods of the disclosureare employed in the treatment or prevention of diarrhea. There aremultiple diarrhea causing organisms including viruses, bacteria,parasites and protozoa. The primary causes of bacterial infection, forexample in India, include Escherichia coli spp., Enterotoxigenic E.coli, Entero-adherent E. coli. Auromonas spp., Camphylobacter jejuni,Shigella spp., Vibrio spp., Vibrio cholera O1, Vibrio parahaemolyticus,Salmonella spp., Staphylococcus aureus, Clostridium difficile,Clostridium perfringens, and Yersinia enterocolitica. Secondary causesinclude Clostridium difficile (toxin A or B), The primary cause of viraldiarrhea is infection by Rotavirus; although Calcivirus, Astrovirus,Norwalk virus, and Adenovirus are also known to cause diarrhea.Secondary causes of viral diarrhea include enteric adenovirus, herpessimplex virus and viral hepatitis. (John B. Sullivan and Gary R.Krieger, Clinical Environmental Health and Toxic Exposures, 2nd Ed.,Lippincott Williams & Wilkins, 2001, page 1040).

There are also known to be regional and seasonal differences inprevalence. For example, in Pranam, India, one study reported rotavirusaccounted for an average 15-25% of childhood cases of diarrhea.Enterotoxigenic E. coli was responsible for 10 to 20% of total diarrheacases, with Enteropathogenic E. coli causing about 1 to 5% of cases.Camphylobacter jejuni infection caused about 10 to 15%, and Shigellacaused an estimated 5 to 15% of cases of childhood diarrhea. Vibriocholera caused about 5 to 10% of cases. Salmonella (non-typhoid) causedabout 1 to 5% of cases. Protozoan infection was caused by primarily byCryptosporidium (5-15%). No pathogenic cause was identified in about 20to 30% of cases. (Fricker, Children in the Tropics, Putting an end todiarrheal diseases, 1993-No. 204: 1-66).

Different regions within India ascribe bacterial cases of childhooddiarrhea to different pathogens with different degree of prevalence. Forexample a study in Orissa, India found, among 866 culture-positivesamples that E. coli sp. (75.5%), pathogenic E. coli (13.2%), Aeromonasspp. (2%), Shigella spp. (4.5%), Vibrio cholera O1 (17.3%), V. choleraO139 (1%) and Salmonella spp. (0.7%),find-health-articles.com/rec_pub_18806340-incidence.

Due to the wide variety of etiology, an effective, broad spectrum,economical and safe method of treating undifferentiated diarrhea isdesired. A majority of childhood diarrhea cases seem to be caused bybacterial and viral infection, but an alternative to antibiotics andantiviral agents is desirable.

A. Compositions

One aspect of the disclosure involves composition useful in thetreatment, prevention or management of microbial flora. In embodiments,the compositions are useful for treating pathogenic infections, inparticular of the gastrointestinal tract.

In embodiments, the disclosure provides a composition comprising:

a) a non-neonate effective amount of at least one specific bindingmolecule, or fragment thereof obtained from an animal and thatspecifically binds to an antigen, wherein the specific binding moleculeis selected from an immunoglobulin, antibody, peptide, variablelymphocyte receptor, transfer factor, and a mixture thereof; and,

b) a carrier matrix comprising at least two components obtained from anonhuman animal selected from the group consisting of enzymes,lactoferrin, trnasferrin, nonspecific immunoglobulins, cytokines, whiteblood cells, complement components, interferons, and fibronectin,wherein the at least one specific binding molecule and the at least twocomponents of the carrier matrix are obtained from different animals.

Specific Binding Molecules

The compositions and methods of the disclosure provide specific bindingmolecules or fragments thereof obtained from an animal and thatspecifically bind to an antigen. A specific binding molecule includes anantibody, an antibody fragment, a peptide, a variable lymphocytereceptor, a transfer factor, and a mixture thereof.

Antibodies

Antibodies, immunoglobulins, and other biological immune factors(referred to collectively as antibodies), both natural and theirsynthetic analogues, are known therapeutic agents in humans and animals.

Antibodies operate by binding (via non-covalent forces) between theantigen-combining site on the antibody and a portion of the antigencalled the antigenic determinant or epitope. Antibodies are capable ofhigh degrees of specificity. For example, the field of monoclonalantibodies has developed largely under the impetus of producing evermore specific and precise binding characteristics. However, this highspecificity can lead to excessively limited binding attributes, whereagents or antigens that are functionally identical do not reactidentically with the immunoreagent or immunotherapeutic.Cross-reactivity on the other hand, usually considered an error orfailure, is the reaction between an antigen and an antibody that wasgenerated against a similar but different antigen. Controlledcross-reactivity may constructively be used to broaden the binding rangeof the antibody.

One embodiment of the present disclosure is based on a method to createa targeted antibody-based formulation embedded or subsumed within acarrier matrix, where the antibodies use a controlled form ofcross-reactivity to multiple clusters of related target antigens, andwhere the carrier matrix contains support and cofactors that enhance theeffect of the antibodies. The utility of such antibody/matrixformulations may include providing broad-spectrum therapeuticinterventions under conditions where the class of causative agent, butnot the precise or specific causative agent is known or suspected orunder circumstances where multiple (mixed) causative agents are active.A novel approach to the use of antibodies in this manner has beendeveloped, that takes advantage of both the specificity andcross-reactive attributes of antibodies, and then further utilizes thecomponents within the carrier matrix to generate a multi-component insitu immune response. In this embodiment, antibodies are designed tobind to all of several closely related epitopes that represent astructurally related cluster of antigens. These antigens may differmarkedly in other respects, and may originate from diverse sources,organisms, or species.

For the purposes of this disclosure, antibodies may be eithermonoclonal, polyclonal derived from any animal, fragments, chimeric,humanized or any other form, and antibodies may be of any isotype: forexample IgA, IgD, IgE, IgG and IgM (placental mammals), IgY (chicken),or others, may be a bispecific or bifunctional, or multispecific ormultifunctional antibody or fragment thereof. In embodiments, thespecific binding molecule can be selected from one of three maincategories: mammalian monoclonal antibodies, mammalian polyclonalantibodies, and avian polyclonal antibodies; or any fragments derivedtherefrom that retain the ability to bind to the pathogenic component.

One embodiment of this invention is its use in the production of a broadspectrum therapeutic. One method for producing this type of reactiveformulation involves the production of polyclonal antibodies harvestedfrom an appropriately immunized animal, and where such antibodies arethen embedded in a carrier matrix. Polyclonal antibodies (or antisera)are antibodies that are derived from from different B cell lines. Theyare typically harvested en-mass from the serum, milk, colostrum, eggs,or biological fluids of an immunized animal. They are a mixture ofimmunoglobulin molecules secreted against a specific antigen, or groupof antigens, recognizing a range of different epitopes. It is possibleto have multiple antibodies for a single antigen (binding to differentepitopes) or for a single antibody to bind to multiple antigens due tocross-reactivity. The polyclonal antibodies can be obtained fromanimals, such as cattle, sheep, horses, goats, swine, rabbits, chickens,ducks, geese, or turkeys that have been vaccinated or inoculated withantigens derived from target components. The antibodies can be harvestedfrom, for example, tissue, serum, milk or eggs produced by, or derived,from the inoculated animal. This is in contrast to monoclonalantibodies, which are identical and monospecific; being produced by onetype of immune cell that are all clones of a single parent cell.

The antibodies used in this invention may be collected from serum,plasma, colostrum, milk, eggs, or other suitable biologically derivedfluid, or from cell culture media, supernatant, etc. The antibodies usedin this invention may be treated in any suitable manner to prepare forformulation and use, including but not limited to separations,plasmapheresis, drying processes, lyophilization, pasteurization, andpreservation methods. The antibodies used in this invention may betreated, concentrated, separated, or purified in various ways dependingupon their final intended use.

By altering the mix of antibodies to those appropriate to variousembodiments, the disclosure provides compositions and methodsappropriate for treating or preventing other gastrointestinal infectionssuch as cholera, C. difficile, dysentery, Salmonella typhi (typhoidfever), and H. pylori (peptic ulcers).

In one embodiment, antibodies are preferably raised in animals by, e.g.,multiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and optionally an adjuvant. In one aspect, it may beuseful to conjugate the relevant antigen (especially when syntheticpeptides are used) to a protein that is immunogenic in the species to beimmunized. For example, the antigen can be conjugated to keyhole limpethemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybeantrypsin inhibitor, using a bifunctional or derivatizing agent, e.g.,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R N═C═NR, where R and Rare different alkyl groups. Animals are immunized against the antigen,immunogenic conjugates, or derivatives as described herein. In otherembodiments, the antibodies may be synthetic or semisynthetic, forexample, as are obtained in a phage display library, or prepared ashumanized or chimeric antibodies.

Birds (such as laying-hens) are highly cost-effective as producers ofantibodies compared with mammals traditionally used for such production.Avian antibodies have biochemical advantages over mammalian antibodies.Immunologic differences between mammals and birds result in increasedsensitivity and decreased background in immunological assays; as well ashigh specificity and lack of complementary immune effects whenadministered to mammalian subjects. In contrast to mammalian antibodies,avian antibodies do not activate the human complement system through theprimary or classical pathway nor will they react with rheumatoidfactors, human anti-mouse IgG antibodies, staphylococcal proteins A orG, or bacterial and human Fc receptors. Avian antibodies can howeveractivate the non-inflammatory alternative pathway. Thus avian antibodiesoffer many advantages over mammalian antibodies.

In a preferred embodiment, the specific molecules are polyclonalantibodies prepared in eggs of hens inoculated with one of or a mixtureof pathogenic components. Various preparations of specific antigens canalso be employed for inoculation. After inoculation, the hen produceseggs containing substantial quantities of specific IgY immunoglobulin inthe yolk, as well as small amounts of IgM and IgA immunoglobulins in thealbumin. Therefore eggs are an excellent source for large quantities ofeconomically produced, highly specific and stable antibodies. In oneembodiment, chickens are used to produce avian antibody; however,turkeys, ducks, geese, ostriches, etc. may alternatively be used. In oneaspect, hens are inoculated by any method known in the art, as describedherein. For example, the antigen may be injected intramuscularly orsubcutaneously. The preferred muscle for injection in an avian is thebreast muscle. Other methods of administration that can be used includesubcutaneous injection, intravenous injection, intraperitonealinjection, intradermal, rectal suppository, aerosol or oraladministration.

The specific immune state is preferably induced and maintained in thetarget animal by immunization and repeated booster administrations of anappropriate dosage at fixed time intervals. The time intervals arepreferably 1-8 week intervals over a period of 1-12 months. Dosage isselected between about 0.01-5 milligrams of the antigen. In one aspect,the dosage is 0.01 mg to 1.0 mg of antigen per inoculation, preferably100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg or 750 mg antigen perinoculation of a hen chicken. The total number of vaccinations can beselected from 1, 2, 3, 4, 5, or 6 in a 12 month period. Typically, afirst inoculation is performed on day 1, with booster vaccinations onday 10, and day 20. The hen chicken can be re-vaccinated as needed bymonitoring the specific antibody concentration, or titer, in the eggsby, e.g., ELISA. A typical subcutaneous dosage volume for a hen chickenis selected from between about 0.2 to 1.0 mL, 0.3 to 0.7 mL, or 0.5 mL.However, it is essential that the booster administrations do not lead toimmune tolerance. Such processes are well known in the art.

It is possible to use other inoculation maintenance procedures orcombination of procedures, such as, for example, intramuscular injectionfor primary immunization and intravenous injection for boosterinjections. Further procedures include simultaneously administeringmicroencapsulated and liquid immunogen, or intramuscular injection forprimary immunization, and booster dosages by oral administration orparenteral administration by microencapsulation means. Severalcombinations of primary and booster immunization are known to thoseskilled in the art.

Adjuvants, also known as pharmaceutical carriers, or functionalequivalents hereof may be included in the immunization solution/vaccinecomposition to enhance the specific immune response of the animal. Alarge number of adjuvants have been described and used for thegeneration of antibodies in laboratory animals, such as mouse, rats,rabbits and chickens. In such setting the tolerance of side effects israther high as the main aim is to obtain a strong antibody response.

Adjuvants pertaining to the present disclosure may be grouped accordingto their origin, be it mineral, bacterial, plant, synthetic, or hostproduct. The first group under this classification is the mineraladjuvants, such as aluminum compounds. Antigens precipitated withaluminum salts or antigens mixed with or adsorbed to performed aluminumcompounds have been used extensively to augment immune responses inanimals and humans. In one embodiment, the adjuvant in the immunizationcomposition is from a bacterial origin. Adjuvants with bacterial originscan be purified and synthesized (e.g. muramyl dipeptides, lipid A) andhost mediators have been cloned (Interleukin 1 and 2). Known chemicalpurification of several adjuvants of active components of bacterialorigin includes: Bordetella pertussis, Mycobacterium tuberculosis,lipopoly-saccharide, Freund's Complete Adjuvant (FCA) and Freund'sIncomplete Adjuvant (Difco Laboratories, Detroit, Mich.) and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.). In a specificaspect, Freund's Complete Adjuvant or Freund's Incomplete Adjuvant isemployed in the immunization compositions of the disclosure.Additionally suitable adjuvants in accordance with the present inventionare e.g., Titermax Classical adjuvant (SIGMA-ALDRICH), ISCOMS, Quil A,ALUN, see U.S. Pat. Nos. 5,876,735 and 5,554,372, Lipid A derivatives,choleratoxin derivatives, HSP derivatives, LPS derivatives, syntheticpeptide matrixes, GMDP, and other as well as combined withimmunostimulants (U.S. Pat. No. 5,876,735). B. pertussis is of interestas an adjuvant in the context of the present invention due to itsability to modulate cell-mediated immunity through action onT-lymphocyte populations. Freund's Complete Adjuvant is the standard inmost experimental studies. Mineral oil may be added to the vaccinationcomposition in order to protect the antigen from rapid catabolism.

Many other types of materials can be used as adjuvants in immunogenic orimmunization compositions according to the present disclosure. Theyinclude plant products such as saponin, animal products such as chitinand numerous synthetic chemicals.

Chickens immunized by the intramuscular route can produce high specificantibody levels in their eggs by day 28 after immunization and continueproducing specific antibodies during more than 200 days making antibodypreparations available in a short period of time, e.g. less than 4-5weeks. Eggs contain IgY antibody concentrations of from up to about 50to about 100 mg per egg. Over 100 mg of purified IgY can be obtainedfrom a single egg. The percentage of antigen specific antibodies in oneegg yolk can be up to about 2% to 10%. (daSilva et al., IgY: A promisingantibody for use in immunodiagnostic and in immunotherapy. VeterinaryImmunol. Immunopath., 135(2010):173-180). One chicken of a highegg-laying strain can produce around 20 eggs per month. Eggs weigh fromabout 33 to about 77 grams, with about 10.5% of the whole egg due toshell. The yolk is about 31% of the weight of the whole egg. Upondrying, about 1 kg of dried whole egg powder can be produced from 72eggs. Therefore, in this calculation, one egg can return about 13.9 gdried whole egg. In another aspect, one immune egg can return from 10 gto about 15 g dried whole egg. In another aspect, the immune eggs of thedisclosure are from 40 to 55 mL per egg with about 1-2 mg/mL total IgYper egg. In another aspect, immune eggs of the disclosure contain about0.01 mg/mL to 0.05 mg/mL specific IgY per egg. Therefore, in one aspectafter processing, one dried whole immune egg contains about 80 to 110 mgtotal IgY and about 6 to 10 mg of total mixed antigen-specific IgY,e.g., from a chicken immunized with, for example a mixed antigenpreparation.

It can be determined whether the vaccine has elicited an immune responsein the egg-producing animal through a number of methods known to thosehaving skill in the art of immunology. Examples of these includeenzyme-linked immunosorbent assays (ELISA), tests for the presence ofantibodies to the stimulating antigens, and tests designed to evaluatethe ability of immune cells from the host to respond to the antigen. Theminimum dosage of immunogen necessary to induce an immune responsedepends on the vaccination procedure used, including the type ofadjuvants and formulation of immunogen(s) used as well as the type ofegg-producing animal used as the host.

In one embodiment, hen chickens suitable for the commercial productionof eggs are employed in the production of polyclonal antibodies. Anybreed of chicken appropriate for egg production can be employed. Forexample, Rhode Island Reds, White Leghorns, Brown Leghorns, LohmannBrown hens, sex-linked hybrid crosses, or other breeds suited to largeegg size, high volume egg production and ease of handling can beselected. In one aspect, chickens are inoculated as chicks as forstandard diseases (e.g. Salmonella, avian influenza, or Newcastle virusetc.). In one aspect, chickens of any age can be inoculated. Hens whichare about to reach laying age, about 15-19 weeks for chickens, or anypreselected time before or thereafter, are inoculated on a schedulepredetermined by the amount and timing of final product to result in asteady continuous production stream. Typically, after a suitable periodof isolation and acclimatization of about 2 to 4 weeks, each group willenter into an inoculation program using various antigens or immunizationcompositions comprising specific antigens to which an antibody isdesired.

In one embodiment, the eggs are collected from inoculated chickens andprocessed as whole eggs. Eggs are stored under refrigeration conditionsuntil enough are collected to prepare a batch. Batches of eggs frompredetermined groups of chickens are cracked, the contents are separatedfrom the shells and mixed and preferably pasteurized to eliminatepotential contamination from pathogenic microorganisms from the chicken.

In one aspect, the immune egg products are pasteurized. Egg products areprocessed in sanitary facilities. Shell eggs are processed into immuneegg product by automated equipment that removes the shell eggs fromflats, washes and sanitizes the shells, breaks the eggs. Optionally, thewhites are separated from the yolks. The liquid egg product isoptionally filtered, optionally mixed with other ingredients, and isthen chilled prior to additional processing. The resulting egg productsliquid then receives a lethality treatment such as pasteurization or isheated in the dried form. In the U.S., the 1970 Egg Products InspectionAct (EPIA) requires that all egg products distributed for consumption bepasteurized.

Following pasteurization, the total egg content is dried using standardcommercial methods, such as spray drying using ambient or hot air,thermal drying, freeze drying, or lyophilization. In one aspect, anappropriate method of drying the pasteurized liquid egg minimizes damageto the antibodies and molecular components in the egg, resulting in aproduct that has a high nutrient value and is capable of conferringpassive protection.

In one aspect, the dried egg is tested to determine overall titer orantibody level. Standard test procedures are used, such as ELISA, FIA(fluorescent immunoassay), RIA (radioimmunoassay), or the like. Inanother aspect, the batch is blended with batches from groups ofchickens at other average production levels resulting in a lotcontaining a standardized amount of antibodies. The dried egg containingspecific polyclonal antibodies may be stored in an airtight container atroom temperature prior to formulation into the compositions of thedisclosure. In embodiments, the dried egg material is used as a wholeegg and is not separated out. In embodiments, the whole dried eggmaterial contains at least 5 mg per egg of specific IgY.

In another embodiment, IgY is isolated. The first step in the isolationof IgY is to separate the water-soluble proteins from lipoproteins.Water-soluble proteins constitute 42.4% of the total proteins in eggyolk (Osuga et al., “Egg Proteins: In Food Proteins, J. R. Whitaker andS. R. Tannenbaum eds., AVI Pub. Co., Westport, Conn. (1977)).

Many methods have been used for the isolation and purification ofimmunoglobulins from egg yolk (Martin et al., Can J. Biochem. Physiol.35:241 (1957); Martin et al., Can. J. Biochem Physiol. 36:153 (1958);Jensenius et al., J. Immunol. Methods 46:63 (1981); Bade et al., J.Immunol. Methods 72:421 (1984); Polson et al., Immunol. Invest. 14:323(1985); Hassl et al., J. Immunol. Methods 110:225 (1988)). Hatta et al.(Agric. Biol. Chem. 54:2531 (1990)) used food-grade natural gums (e.g.,carrageenan) to remove yolk lipoprotein as a precipitate and to recoverIgY in the water-soluble fraction from egg yolk. Methods for recoveringantibodies from chicken egg yolk are well known in the art. Severalmethods can be used for the extraction of IgY from egg yolk, andcommercial extraction kits are available (van Regenmortel, M. H. V.(1993). Eggs as protein and antibody factories. In Proceedings of theEuropean Symposium on the Quality of Poultry Meat, pp. 257-263. Tours,France: INRA).

In another embodiment, the steric specific binding molecule may be amonoclonal antibody specific for a pathogenic component.

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature. 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridomamethod, a mouse or other appropriate host animal, such as a hamster, isimmunized as described above to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. After immunization, lymphocytes are isolated andthen fused with a myeloma cell line using a suitable fusing agent, suchas polyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (MA) or enzyme-linkedimmunosorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysisdescribed in Munson et al., Anal. Biochem. 107:220 (1980).

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.

Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pl{acute over (υ)}ckthun. Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al. Nature. 348:552-554 (1990). Clackson etal. Nature. 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology. 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Antigens for Immunization to Prepare a Specific Binding Protein

The antigens selected for immunization can be bacterial, viral,protozoal, fungal, parasitic, cellular, or any other substances to whichthe immune system of an animal will respond. In one aspect, theimmunogenicity of the antigens is enhanced by use of an adjuvant.

In one aspect, the animal is inoculated with the pathogenic components,antigens, or immunogens in a vaccination composition, inoculant orvaccine. In one aspect, the pathogenic components or specific antigenscan be obtained or derived from commercial sources such as the AmericanType Culture Collection (ATCC). In another aspect, the pathogeniccomponents can be isolated from a wild type strain. In another aspect,the pathogenic components or undesirable strains are present in a mixedantigen preparation. Any antigens or combination of antigens derivedfrom various undesirable strains or pathogenic components can beemployed in the immunization composition.

In one aspect, the inoculant, antigen or immunogen is selected to acommon or preserved component or region of the targeted antigen cluster,while ignoring the variable or distinguishing components or regions ofthe individual members of the cluster of related antigens. The methodinvolves the preparation of an appropriate immunogen withcharacteristics that elicit the production of antibodies that arecross-reactive to desired instances of that epitope, but which are notreactive to other epitopes, and the inoculation or exposure of theproducing cells or organism to that immunogen so as to cause theproduction of antibodies, with the resultant antibodies being embeddedwithin the suitable carrier matrix for administration. Formulations ofthis type may be developed that use admixtures of antibodies producedaccording to this method to provide broad coverage of more than onecluster of target antigens. For example, in the case where two clustersof unrelated antigens are associated with a disease or condition, and itis desirable to create a single formulation to address this disease orcondition, an admixture of two antibodies, immunoglobulins, orbiological immune factors may be prepared using this method thatsimultaneously provides two broad domains of reactivity. One example ofthis embodiment is for the production of antitoxin antibodies that arespecifically reactive to clusters of structurally related toxins.

In one embodiment, this approach is used to make a broadly reactiveantibody to lipopolysaccharide (LPS) (endotoxin) from any Gram-negativebacteria (Escherichia coli, Salmonella, Shigella, and otherEnterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, and others), or for example a broadly reactiveantibody to AB5 toxins (including Campylobacter enterotoxin, choleratoxin, heat-labile enterotoxins (LT and LT-II) (E. coli), pertussistoxin, shiga toxin (Shigella), shiga-like toxin (or verotoxin)).

In a preferred aspect, these example anti-toxin antibodies have effectwithout regard to the species originating the toxin. In another aspect,the antibodies produced are neutralizing antibodies, capable ofneutralizing or inactivating the biological activity of the targettoxins. Such a broad-spectrum neutralizing antibody could be used tointervene in pathology cases (for example certain types of diarrhea)where the toxin mediating the symptoms was one of the cluster targeted(in these examples, AB5 or LPS), without requiring knowledge of whichorganism was causative. Further, if an admixture was prepared containingboth the anti AB5 antibody and the anti LPS antibody in clinicallyeffective amounts, the formulation could be used to intervene in casewhere the active toxin was either AB5 or LPS or both.

This method can be extended to include any number of (in this example)toxin clusters, and to include broad-spectrum neutralizing antibodies tomediators of other toxin-like reactions (for example viral toxin-likephenomena), to create a broadly applicable intervention (in this exampleto) diarrhea where symptoms and pathology can be managed withoutknowledge of the infectious causes, or where there are multipleinfectious causes. In one aspect, the disclosure provides a compositioncomprising a synergistic combination of anti-toxin antibodies combinedin a carrier matrix.

In some embodiments, the methods and compositions of the invention areused for a variety of pathogens or agents including, without limitation,cholera toxin (Vibrio cholera), E. coli (including enterotoxigenic(ETEC)), Shigella, Salmonella, Campylobacter, Clostridium difficile,parasites (e.g., Giardia, Entamoeba histolytica, Cryptosporidiosis,Cyclospora), and diarrheal viruses (e.g., rotavirus).

After entering the gastrointestinal tract many pathogens, including butnot limited to bacteria such as E. coli, bind (adhere) to epithelial,mucosal, or other tissue and become embedded in gastrointestinal tracttissue, such as the wall of the intestine. After binding to tissue inthe gastrointestinal tract the pathogens replicate, causing an increasein toxin concentrations, either directly from production or indirectlyfrom increased lysing of pathogen cells by immune system action.Inhibiting the ability of pathogens to bind to the gastrointestinaltract tissue promotes a more effective mobilization of the pathogens,digestion and excretion before colonies of sufficient size to causelesions and other symptoms are formed. By blocking the class ofreceptors and ligands on the pathogen that would be used to adhere tothe gastrointestinal tract, including but not limited to adhesins,cadherins, cilia, fimbrillae, and/or viral adhesion structures, adhesionto gastrointestinal tract tissue can be prevented or minimized,ultimately resulting in substantially decreased pathology from pathogensthat utilize this mode of action.

In various embodiments, the pathogen is selected from one or acombination of human or veterinary, enteric or gastrointestinal,pathogens causing gastroenteritis. In various aspect, the pathogen isselected from the group consisting of: Campylobacter jejuni, Salmonella,Salmonella typhimurium, Salmonella enterica serovar Typhi, Shigelladystenteriae, Plesiomonas shigelloides, Escherichia coli [including(EPEC) enteropathogenic E. coli, (ETEC) enterotoxigenic E. coli,(EaggEC) enteroaggregative E. coli, (EIEC) enteroinvasive E. coli, and(EHEC) haemorrhagic E. coli], Yersinia enterocolitica, Vibrio choleraeO1, Vibrio O139, Non-O1 Vibrios, Vibrio parahaemolyticus, Aeromonashydrophila, Clostridium perfiingens, Clostridium difficile,enterohepatic Helicobacter (including Helicobacter pylori),Staphylococcus aureus, Klebsiella, rotavirus, coronavirus, norovirus,calicivirus, enteric adenovirus, cytomegalovirus, and astrovirus. Inanother aspect, the pathogen related toxin includes an endotoxin orexotoxin. In another aspect, the pathogen related adhesion elementincludes adhesins, cadherins, cilia, fimbrillae, a viral adhesionstructure, or a combination thereof.

In various specific aspects the pathogenic components, immunogens orantigens can be derived from, e.g., Rotavirus, Corona virus; ClostridiumPerfringens Type C; Escherichia coli (cellular); Enterotoxigenic strainsof, and Enterotoxins from, E. coli; any bacteria having K99, K88, 987P,or F41 pili adherence factor antigen; endotoxin (or LPS) caused by E.coli and Salmonella typhimurium (gram negative bacteria, generally). Ina particular aspect, hens are inoculated with antigens or toxins derivedfrom one, two, three, four, five, six, seven, or eight, or a number ofpathogenic microorganisms.

In one aspect, the immune response is more potent when the distancebetween the antigen source and the immune system of the vaccinatedanimal increases.

In a specific embodiment, a first flock of chickens is inoculated with afirst one mixed antigenic preparation. In one aspect, a second flock ofchickens is inoculated with a second mixed antigenic preparationcontaining a different set of antigens than the first. In anotheraspect, a third flock of chickens is inoculated with a third mixedantigenic preparation. In a further aspect, a fourth flock of chickensis inoculated with a fourth mixed antigenic preparation. While not meantto limit the scope of the invention, it is believed to be advantageousto immunize different flocks with different antigens in order to avoidantigen overload.

Eggs from each flock are collected, optionally titered as to specificand/or total IgY, optionally isolated and/or purified, and processedseparately to prepare a dry powder. In another aspect, dry powdered eggsfrom the first and second; first, second and third; or first, second,third and fourth flocks are blended, or packaged, with a carrier matrixto prepare a composition of the disclosure. In one aspect, a firstantigenic preparation comprises bovine rotavirus (serotypes G6 and G10),bovine coronavirus, enterotoxigenic strains of Escherichia coli havingthe K99 pili adherence factor, and Clostridium perfringens type C. Themixed antigenic preparation can is optionally adjuvanted to enhance theimmune response.

In another aspect, a second antigenic preparation comprises beta toxinproduced by Clostridium perfringens type C and enterotoxigenic strainsof Escherichia coli producing heat-labile toxin or having the K99, K88,987P, or F41 adherence factors.

In one aspect, a third antigenic preparation comprises E. coli andSalmonella typhimurium. JVAC reduces the incidence and severity ofendotoxemia caused by E. coli and Salmonella typhimurium. Commonlyassociated with their endotoxins are Coliform Mastitis and othergram-negative diseases associated with Endotoxemia.

In another aspect, the antigens are prepared by any means known in theart. For example, cells from a wild type source, such as an animalsuffering from, e.g., E. coli diarrhea. The isolate cells can becultured in, e.g., Trypticase Soy Broth (TSB) at 37° C. overnight andconcentrated by centrifugation. The resulting pellet can be re-suspendedwith 0.4% formaldehyde in PBS buffer and incubated at 37° C. forinactivation. Formaldehyde can be removed by centrifugation. The pelletcan be resuspended in PBS and used as antigen. In one aspect, theantigens are emulsified with an equal volume of adjuvant prior toinoculation.

In another embodiment, the antigens are selected from those pathogenicorganisms causing conjunctivitis. Known causative pathogens aredescribed in US 2008/0031903, Gambotto et al., which is incorporatedherein by reference.

Epidemic Keratoconjuctivitis (EKC) is a debilitating infectious diseaseof the eye that is seen all over the world. The disease is caused mostlyby adenoviruses especially serotype 8, 19 and 37. Serotype 3, 4 and 11were also implicated in some EKC epidemics. The disease affects all agegroups, is highly contagious and spreads quickly in schools, schools,swimming pools, pediatric unit and camps. Treatment is presentlysymptomatic as there is no effective treatment. Development of effectiveanti-viral topical agent is desirable to treat the disease and preventepidemic.

Conjunctivitis also can be caused by a number of additional bacterial,viral, fungal and protozoa agents, including, but not limited to: S.aureus, S. pneumoniae, H. influenzae, Neisseria gonorrhoeae, Chlamydiatrachomatis, Adenovirus, Herpes Simplex, Herpes zoster virus,Enteroviruses, Fusarium species, Candida species and Acanthamoebaspecies. Certain viral infections, such as adenoviral infections may betreated with antiviral drug products, such as cidofovir. Typically, drugproducts have side effects, such as the ocular and renal side effectsassociated with cidofovir. Other logistical issues arise with drugproducts, including stability, cost of production, etc. As such, aninexpensive, readily-available, well-accepted and stable drug productfor treatment of ocular infections is desirable.

In one aspect, the disclosure provides a composition for the treatmentof conjunctivitis, or pink eye, comprising polyclonal antibodies tothese pathogens combined in a carrier matrix as described below. Theantibodies are produced as described herein.

In another embodiment, the antigens are selected from those pathogenicorganisms causing vaginitis. The infection may be bacterial, fungal(yeast), or parasitic. Bacterial vaginitis can be caused, for example,by Gardnerella spp., Neisseria gonorrhoeae, Chlamydiaceae trachomatis,Mycoplasma spp., Campylobacter jejuni. Parasitic vaginitis can be causedby, e.g., Trichomonas vaginalis. Viral vaginitis can be caused by e.g.,herpes virus type 1 or type 2. Candidal vaginitis is caused by yeastlikefungi Candida. There are more than 170 species of yeastlike fungi isdescribed. C. albicans is the most frequent causative agent of acandidal vaginitis in 85-90% of women. C. glabrata (5-10%), C.tropicalis (3-5%), C. parapsilosis (3-5%) and C. krusei (1-3%) are alsoclinically significant among other species of Candida. Any of thesepathogens may be selected as the antigenic source for polyclonalantibody production as described herein.

Candidal vulvovaginitis is frequently caused by a number of predisposingfactors, such as long and uncontrolled using of antibiotics,corticosteroids, cytostatics, oral contraceptives, radiation therapy,serious infectious disease, endocrine disorder, immunodeficiency state,etc. Prescription of broad spectrum antibiotics suppresses not onlypathogenic bacteria, but also mucous vaginas saprophytes: lactobacilliand bifidobacteria. As a result vaginal pH raises (towards to alkalinerange), and disturbance of self-cleaning processes occurs. Besides,Candida is able to use some antibiotics as nutrient substrates. Thusfavorable conditions for active overgrowth of Candida arises in femalegenital organs. In one aspect, the disclosure provides a composition forthe treatment of vaginitis comprising polyclonal antibodies to one ormore of the described pathogens combined in a carrier matrix asdescribed below.

In a specific aspect, the composition of the disclosure comprising amixture of specific polyclonal antibodies in a carrier matrix provides abroad spectrum method of treating bacterial, viral, fungal or parasiticvaginitis. In another aspect, the compositions of the disclosure can beused to treat undifferentiated vaginitis in a subject in need thereof

Other Specific Binding Molecules

The compositions and methods of the disclosure include other specificbinding molecules including transfer factors, variable lymphocytereceptors and cell receptors. A transfer factor is an immune molecule ofapproximately 5000 Daltons, made up of amino acids, that causeantigen-specific cell-mediated immunity, primarily delayedhypersensitivity and the production of lymphokines, as well as bindingto the antigens themselves. (Kirkpatrick 1993, Structural nature andfunctions of transfer factors. Ann. N.Y. Acad. Sci. 685:362-368.)Variable lymphocyte receptors are lymphocyte-derived moleculesdiscovered in jawless vertebrates such as the lamprey and hagfish. Theseanimals possess a large array of variable lymphocyte receptors that areproduced from only a small number of genes and that bind to pathogenicantigens in a similar way to antibodies, and with the same degree ofspecificity. (Alder et al., 2005, Diversity and function of adaptiveimmune receptors in a jawless vertebrate. Science, 310(5756):1970-1973).

Carrier Matrix

The disclosure provides compositions for the treatment or prophylaxis ofpathogenic infection in a subject. The compositions comprise specificbinding molecules, such as polyclonal antibodies, combined with acarrier matrix. While not meant to limit the scope of the invention, thecarrier matrix serves a dual purpose. First, to protect the antibodiesin their intended functional environment, for example, upon oraladministration, and within the gastrointestinal tract of the non-neonatesubject; and further to provide components, e.g., components of theinnate immune system, to react synergistically with the antibodies inthe management of an infection.

The term “carrier matrix”, or protective/reactive matrix, refers to anysubstrate, compound, formulation, or supplemental admixture (whethernatural or synthetic) containing elements, co-factors, or othercomponents in appropriate ratios and concentrations so as to supplyelements required to propagate, promote, support, or enhance an in situimmune-type response, cascade, or reaction. These elements may variouslypromote cleavage and maturation reactions, the formation of assembliesand complexes, depletion and adsorption functions, supply essentialelements, biologics, or compounds, and provide protective functions foractive elements or components. A carrier matrix may or may not containendogenous antibodies (immune factors), which may or may not be specificto targeted antigens.

In one embodiment, the carrier matrix the matrix is selected from, orderived from, serum, plasma, colostrum, milk, saliva, lymph fluid,mucous, or lachrymal fluid derived from a non-human mammal.

An example of a naturally occurring carrier matrix is colostrum.Colostrum has evolved naturally in mammals specifically to deliver itscomponents to neonates to and through the gastrointestinal tract in avery concentrated low-volume form. Colostrum, or “first milk”, isproduced by mammals immediately postpartum. The antibodies and cofactorsare passed to the neonate from the mother and provide the firstprotection against pathogens. Growth factors also stimulate thedevelopment and repair of the gut.

Colostrum contains a host of immuno-complimentary factors. They includeinterferons, immunglobulins (including IgG and secretory IgA),polymorphonuclear leukocytes, macrophages, and lymphocytes. Colostrumalso contains proline-rich polypeptide, or PRP, a T-cell activator.Colostrum is known to be high in immunoglobulin content compared tomilk. Colostrum is known to contain antibodies such as IgA, IgG, and IgMin mammals. IgA is absorbed through the intestinal epithelium, travelsthrough the blood, and is secreted onto other Type 1 mucosal surfaces.Bovine Colostrum is noted to be anywhere from 6% to 20% immunoglobulin;primarily IgG₁ and IgG₂. In one aspect, whole bovine colostrum is usedas the carrier matrix.

Colostrum also helps to regulate the intestinal environment, renderingit hostile to foreign pathogens. Colostrum contains lactoferrin, aniron-binding protein that prevents bacteria and viruses from obtainingiron necessary for replication. Colostrum also selectively fertilizescertain probiotic species that in turn help to ward off infection. It isthe only natural source of two major growth factors, Transforming GrowthFactors (TGF) alpha and beta, as well as a source of Insulin-GrowthFactors 1 and 2. These factors promote tissue repair and development.Colostrum is also a source of Hepatocyte Growth Factor, which stimulatesthe growth and expansion of intestinal wall cells. Colostrum isnaturally designed to serve as a carrier matrix within agastrointestinal environment. Synthetic versions of a carrier matrix arealso included in this disclosure. Carrier matrices that are composed ofboth natural and synthetic components are also included within thedisclosure.

Colostrum is very rich in proteins, vitamin A, and sodium chloride, butcontains lower amounts of carbohydrates, lipids, and potassium thannormal milk. The most pertinent bioactive components in colostrum aregrowth factors and antimicrobial factors. The antibodies in colostrumprovide passive immunity, while growth factors stimulate the developmentof the gut. They are passed to the neonate and provide the firstprotection against pathogens. The passive immunity from the mother getstransferred to the newborn.

Newborns have very small digestive systems, and colostrum delivers itsnutrients in a very concentrated low-volume form. The gastrointestinaltract of the neonate is particularly receptive to passive transfer ofimmunity from colostrum. At birth gastric pH ranges vary from 6-8 due toresidual amniotic fluid in the stomach. Gastric pH then falls to a pH of1.5 to 3 in 24 to 48 hours. Therefore, The GI conditions of the newbornare conducive to passive immunization. In addition, gastric emptyingtime in neonates and premature infants is prolonged, with adult valuesbeing reached at 6-8 months of age. The antibodies and cofactors incolostrum can, under certain circumstances (e.g., breastfeeding) providea passive immunity to the recipient; this is particularly true for theneonate. The gastrointestinal tract of non-neonatal babies, children,adolescents and healthy adults is a more hostile environment withrespect to oral administration of immunoglobulins.

Other immune components of colostrum include the major components of theinnate immune system, such as lactoferrin, transferrin, lysozyme,lactoperoxidase, complement, and proline-rich polypeptides (PRP). Anumber of cytokines (small messenger peptides that control thefunctioning of the immune system) are found in colostrum as well,including interleukins, tumor necrosis factor, chemokines, and others.Colostrum also contains a number of growth factors, such as insulin-likegrowth factors I, and II, transforming growth factors alpha, beta 1 andbeta 2, fibroblast growth factors, epidermal growth factor,granulocyte-macrophage-stimulating growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, andcolony-stimulating factor-1.

In one aspect, the carrier matrix is comprised of two or more, three ormore, four or more, five or more, or six or more, or seven or morenon-immunoglobulin components of colostrum. In another aspect, thecarrier matrix comprises colostrum that has been processed to remove themajority of immunoglobulins. In embodiments, a carrier matrix comprisesat least two components obtained from a nonhuman animal selected fromthe group consisting of enzymes, lactoferrin, transferrin, nonspecificimmunoglobulins, components of the complement system, cytokines, whiteblood cells, complement components, interferons, and fibronectin,wherein the at least one specific binding molecule and the at least twocomponents of the carrier matrix are obtained from different animals. Inanother aspect, the matrix is comprised of two or more agents selectedfrom lysozyme, phospholipase, defensins, opsonins, proline-richpolypeptides (PRP), beta-lysin, lactoferrin, transferrin, cytokines,interleukins, chemokines, interferons, TNF-alpha, fibronectin,proline-rich polypeptides, insulin growth factor type 1, insulin GrowthFactor type 2, derived platelet growth factor, epidermal growth factor,fibroblast platelet growth factor, transforming growth factor alpha,transforming growth factor beta, nerve growth factor, leptin,leukocytes, white blood cells, phagocytes, macrophages, monocytes,neutrophils, polymorphonuclear cells, and dendritic cells, mast cells,eosinophils, basophils, natural killer (NK) cells, lymphokine activatedkiller (LAK) cells, cationic proteins including defensins, proteolyticenzymes including elastase, cathepsin G, myeloperoxidase, NADPH oxidasecomponents, or a combination thereof. In another aspect, the matrixincludes a mixture of agents from the innate immune system. In apreferred aspect, the carrier matrix is comprised of non-hyperimmunebovine colostrum.

Bovine colostrum is produced by cows for their newborn calves. In manydairy cow herds the calves are not permitted to nurse; rather, they arefed colostrum and later milk from a bottle then a bucket. The colostrumis collected and processed for commercial uses. Various compositionsincluding colostrum and processes for preparing colostrum have beendisclosed in U.S. Pat. Nos. 5,846,569, 6,410,058, 6,475,511, and6,521,277, the contents of which are incorporated by reference in theirentireties. Dried bovine colostrum is commercially available. In onespecific aspect, the carrier matrix is commercial dried bovinecolostrum.

Livestock husbands/breeders commonly bank colostrum from their animals.Colostrum produced on their own premises is considered to be superior tocolostrum from other sources, because it is produced by animals alreadyexposed to (and thus making antibodies to) pathogens occurring on thepremises. Generally, colostrum from animals exposed to relevantpathogens will have superior immunological characteristics.

Bovine colostrum and its components are safe for human consumption,except in the context of intolerance or allergy to lactose or othercomponents. Bovine colostrum from pasture-fed cows containsimmunoglobulins specific to many human pathogens, including Escherichiacoli, Cryptosporidium parvum, Shigella flexneri, Salmonella,Staphylococcus, and rotavirus, depending upon their natural exposure tothese pathogens. Before the development of antibiotics, colostrum wasthe main source of immunoglobulins used to fight infections.

Hyperimmune colostrum represents an attempt to boost the effectivenessof natural bovine colostrum by immunizing cows with a specific pathogen.This approach is promising as antibodies are produced to the specificpathogens or antigens used in the original challenge. However, varyingresponse to antigens, biological variability, and low production yieldof colostrum have limited its clinical and commercial utility.

In one aspect, the disclosure provides a composition comprisingcolostrum that is not hyperimmune colostrum or that does not contain ameasurable or significant amount of antibodies specific for thepathogenic or target antigen components. In another aspect, thedisclosure provides a composition in which the carrier matrix containsvarious components of the innate immune system without a significantamount of either specific or non-specific antibodies.

In one embodiment, the colostrum can be processed to remove the majorityof immunoglobulin, e.g., by absorbing the antibodies onto an affinityresin (e.g. Protein G or Protein A Sepharose; or Protein A or Protein GAgarose) in a batch or column format and retaining the eluate forfurther processing. Immunoglobulin can also be removed by gel filtrationchromatography on Sephadex G-200 or DEAE Sephadex A-25 ion exchangechromatography. (Lloyd and Soulsby, Immunology, The role of IgAimmunoglobulin in the passive transfer of protection to Taeniataeniaeformis in the mouse. 34, 939-945) These processes can be run on acolumn or a batch format by various methods and techniques known in theart.

In one specific embodiment, the carrier matrix includes colostrum. Inone aspect, commercial colostrum is employed as the supportive/reactivematrix. In a preferred aspect, the commercial bovine colostrum is anagglomerated and instantized, pasteurized, full cream, whole colostrumpowder produced from first milking colostrum only. In another aspect,the colostrum is processed at low pressures and low temperatures and isspray dried using indirect steam to maintain maximum bioactivity. Inanother aspect the commercial colostrum is from antibiotic free sources.In another aspect, the colostrum is subjected to microbiologicalanalysis and is found to be negative, or below acceptable levels withrespect to a variety of pathogens. In various other aspects, thecolostrum is assayed for other contaminants such as nitrates, aflatoxin,nitrofuran, dioxins, melamine, and heavy metals and found to be negativeor below specified levels.

In one embodiment, the invention may be composed of colostrums ofseveral hyperimmunized sources, each targeting a different cluster orclass of antigen, where the colostrums are admixed to provide abroad-spectrum antibody formulation.

In another embodiment, the carrier matrix is comprised of areconstituted or artificial mucosal secretion such as tear fluid, nasalor bronchial mucous, cervical mucous, seminal plasma, sweat, bloodplasma or saliva. Body fluids are known to contain several components invarying amounts. (Schenkels et al., Biochemical composition of humansaliva in relation to other fluids, Crit. Rev. Oral Biol. Med., 1995,6(2):161-175). Saliva contain mucins, acidic PRPs, alpha-amylase, basicPRPs, basic PRG, secretory IgA, cystatins, statherin, IgG, extra-parotidglycoprotein (EP-GP), VEGh (a lipocalin), histatins, lysozyme,kallikrein, lactoferrin, lactoperoxidase, haptocorrin,beta-microseminoprotein, IgM, albumin, and Zn-alpha2-glycoprotein. Inone aspect, the carrier matrix comprises two or more, three or more,four or more, five or more, six or more, or seven or more of thecomponents of body fluids.

Tear fluid, or lachrymal fluid, has many of the same components assaliva and has a particularly high concentration of secretory IgA, VEGh,lysozyme, and lactoferrin. In one aspect, artificial lacrimal fluidscontaining salts such as sodium chloride and the like as a mainingredient, or eye drops containing hydroxyethylcellulose, chondroitinsulfate or hyaluronic acid or xanthan gum (U.S. Pat. No. 7,875,271,which is incorporated herein by reference) as known in the art arefortified with two or more, three or more, four or more, five or morecomponents of the body fluids as described and used as a carrier matrixfor purified polyclonal antibodies, as described herein. In one aspect,a composition could be used to treat microbial infections of the eye,such as pink eye.

Cervical mucous contains mucins, alpha-amylase, lysozyme,lactoperoxidase, albumin, and beta-microseminoprotein. The matrix isformed by combination of two or more, three or more, four or more, fiveor more of these components as a carrier matrix in a composition with asteric specific binding molecule, such as anti-bacterial or anti-fungalpolyclonal antibodies, prepared by the methods of the disclosure.

In one aspect, the disclosure provides a composition comprising a gumcapable of fixing water or swellable in water, containingcarboxymethylstarch combined with a cellulose as a permeating agent,which when put into contact with water form almost instantaneously gelsand is readily applicable for vaginal application. Tablets comprisingthe antibody/matrix composition of the disclosure could for examplecomprise carboxymethylstarch and cellulose as described in U.S. Pat. No.4,808,415, which is incorporated herein by reference. In a particularaspect, the antibacterial and antifungal polyclonal antibodies arecombined in the matrix and formulated to provide a broad spectrumtreatment for vaginosis. In one aspect, the composition is used to treata vaginal bacterial infection, such as trichomonas infection, or fungalvaginosis, such as a candida infection.

Saliva is a mucosal secretion present in the oral cavity and produced bysalivary glands. Saliva serves protective functions such as tissuecoating, lubrication, humidification, and remineralization of the teeth.Saliva also serves host defense functions with immunological activity,anti-bacterial, anti-viral and anti-fungal activity. Saliva also servesdigestive activity with digestive enzymes, bolus formation and taste.Saliva contains various proteins such as histatins, and acidicproline-rich proteins that are unique to saliva. Saliva also containsproteins present in other body fluids such as lysozyme, mucins,statherins and immunoglobulins. Saliva contains proteins such as albuminand Zn-alpha-2-glycoprotein that originate in blood plasma. There is aknown therapeutic value of bovine saliva. (Varshney et al., 1997,Therapeutic value of bovine saliva in wound healing: ahistomorphological study., Indian J. Biol. May 1997, 35(5):535-7). Inone aspect, components of saliva could be useful, for example, intoothpaste or mouthwash, or other preparations for oral mucosaladministration.

Bronchial mucous contains mucins, alpha-amylase, basic proline-richpolypeptides (PRPs), cystatins, statherin, EP-GP, lysozyme,beta-microseminoprotein, and albumin. In one aspect, the disclosureprovides a composition comprising a steric specific binding molecule anda carrier matrix comprising two or more, three or more, four or more ofthe components of saliva or bronchial secretions. In one aspect, thecomposition with the carrier matrix is to be packaged in a dry formatwith the steric specific binding molecule, such as anti-Group AStreptococcus polyclonal antibodies prepared according to thedisclosure. In one aspect, the dry formulation is reconstituted, forexample in a saline solution, and administered as a throat spray fortreatment of strep throat.

Other carrier matrices may be prepared to function in other useenvironments, for example for aerosolized (inhaled), ocular, topical, orother preparations.

In a specific embodiment, the specific binding molecule and the carriermatrix are derived from different species. In a further aspect, both thespecific binding molecule and the carrier matrix are derived fromnon-human species. In another aspect, the specific binding molecule isderived from a non-mammalian animal. In another aspect, the carriermatrix is derived from a non-human mammal.

Formulations and Compositions

In one embodiment antibodies are harvested from the plasma, serum, orblood, colostrum, eggs, or other component of an inoculated animal orartificial production system (such as cell culture), then purified ortreated, and added to a carrier matrix such as colostrum. Thecompositions allow are used as a delivery medium for, e.g., oraladministration of the antibody formulation. This approach may provide aneffective way of reliably scaling antibody production for formulation inthis manner, so as to control titer, consistency, and continuousavailability, for commercial use. In one embodiment antibodies areharvested from the eggs of an inoculated animal, and may be purified ortreated or retained in the egg material, and added to bovine colostrums.

There is a clear need for low cost and effective treatments for manygastrointestinal pathogens, and orally administered antibodies arecandidates for this role. In addition to having demonstrated efficacy,orally administered antibodies are typically non-immunogenic. They areconsidered typically well tolerated with no adverse side effectsreported and comparatively no different reactions than a comparableingested food product. Notably several products containing orallyadministered antibody have received GRAS (Generally Recognized as Safe)certification by the FDA.

One embodiment of this invention is a broad spectrum therapeutic orprophylactic antitoxin formulation composed of an admixture ofbroad-spectrum neutralizing antibodies, embedded within a carriermatrix, produced according to this method, for the purposes of allowingfor effective administration across a wide range of unknown orundiagnosed conditions resulting in toxin mediated diarrhea.

One embodiment of this invention is a broad spectrum therapeutic orprophylactic anti-pathogen formulation, embedded within a carriermatrix, containing an admixture of broad-spectrum anti-pathogenantibodies produced according to this method.

One embodiment of this invention is a broad spectrum therapeutic orprophylactic anti-adhesin formulation embedded within a carrier matrix,containing an admixture of broad-spectrum anti-adhesin antibodiesproduced according to this method.

One embodiment of this invention is a broad spectrum therapeutic orprophylactic formulation embedded within a carrier matrix, containing anadmixture of broad-spectrum antitoxin, anti-pathogen, and anti-adhesinantibodies produced according to this method.

One important limitation of using natural food based products is thatpreparations are limited to the results allowed by natural processes.The present invention allows for the selective addition of levels ofspecific antibodies and general immune factors (formulation) that aresignificantly higher than physiological levels that can normally beachieved in nature. The present invention also allows for a weighting ofvarious factors in a manner so as to create greater specificity totargeted diseases, pathogens, or substances.

In one embodiment, the formulation comprising the specific bindingmolecule is a dry solid (egg powder) formulation. The powderedformulation is sealed in airtight packets, optionally layered with aninert gas. The formulation can be stored for extended periods of time atroom temperature, under refrigeration, or frozen temperatures. In otherembodiments, the dried composition is formulated into capsules ortablets for oral administration. In another embodiment, the formulationis compressed into chewable tablets.

Another embodiment of the present invention relates to thepharmaceutical acceptable diluents for formulating the composition,wherein said pharmaceutical acceptable diluents are selected from thegroup consisting of a lactose, mannitol, sorbitol, microcrystallinecellulose, sucrose, sodium citrate, dicalcium phosphate, or any otheringredient of the similar nature alone or in a suitable combinationthereof; binder selected from the group consisting of gum tragacanth,gum acacia, methyl cellulose, gelatin, polyvinyl pyrrolidone, starch orany other ingredient of the similar nature alone or in a suitablecombination thereof; excipients selected from the group consisting ofagar-agar, calcium carbonate, sodium carbonate, silicates, alginic acid,corn starch, potato tapioca starch, primogel or any other ingredient ofthe similar nature alone or in a suitable combination thereof;lubricants selected from the group consisting of a magnesium stearate,calcium stearate or steorotes, talc, solid polyethylene glycols, sodiumlauryl sulfate or any other ingredient of the similar nature alone;glidants selected from the group consisting of colloidal silicon dioxideor any other ingredient of the similar nature alone or in a suitablecombination thereof; a sweetening agent selected from the groupconsisting of such as sucrose, saccharin or any other ingredient of thesimilar nature alone or in a suitable combination thereof; a flavoringagent selected from the group consisting of peppermint, methylsalicylate, orange flavor, vanilla flavor, or any other pharmaceuticallyacceptable flavor alone or in a suitable combination thereof; wettingagents selected from the group consisting of acetyl alcohol, glycerylmonostearate or any other pharmaceutically acceptable wetting agentalone or in a suitable combination thereof; absorbents selected from thegroup consisting of kaolin, bentonite clay or any other pharmaceuticallyacceptable absorbents alone or in a suitable combination thereof;retarding agents selected from the group consisting of wax, paraffin, orany other pharmaceutically acceptable retarding agent alone or in asuitable combination thereof.

In another aspect, the daily dose for the non-neonate human isstandardized by any method of quantifying the specific antibodies. Inone aspect, the dose of the composition is standardized by use of anELISA to evaluate the concentration of specific anti-antigen antibody inthe formulation. In one aspect, one dose of the oral compositioneffective to treat a pathogenic infection contains antigen-specificbinding molecule in an amount from about 0.0001 mg to 20 mg; from 0.001mg to 15 mg; from 0.01 to 10 mg; from 0.05 to 5 mg; from 0.1 to 1 mg ofmixed antigen specific binding molecule.

The term “solid form” refers to a dried form of a specific bindingmolecule, or a dried form of a carrier matrix, or a solid dosage formcomprising both the dried specific binding molecule and the carriermatrix as a powder, compressed tablet, troche, or capsule. In oneaspect, the solid dosage form is intended for oral administration. Inone aspect, the powder is a formulation for suspension. In one aspect,powdered dried immune egg and powdered dried colostrum are packaged inan airtight packet. Immediately prior to oral administration, thecontents of the packet are suspended, or dissolved, in about a liquidand administered orally.

In one aspect, the composition may also be provided in a liquid form foradministration.

In one aspect, one dose contains 1 g, 2 g, 3 g, 4 g, 5 g, 5 g, 6 g, or 7g of dried immune egg and 1 g, 2 g, 3 g, 4 g, 5 g, 5 g, 6 g, or 7 gdried bovine colostrum. In one aspect, one dose of the dried dosage formcontains 3 g dried immune egg product and 4 g dried bovine colostrum. Inone aspect, one dose of the dried dosage form contains 2 g dried immuneegg product and 4 g dried bovine colostrum. In one aspect, one dose ofthe dried dosage form contains 4 g dried immune egg product and 4 gdried bovine colostrum. In another aspect, the contents of a single dosepacket are dissolved in about 2 ounces of water and administered orally.

Formulations for oral use may also be prepared as troches, chewabletablets, or as hard gelatin capsules wherein the active ingredient ismixed with an inert solid diluent (e.g., potato starch, lactose,microcrystalline cellulose, calcium carbonate, calcium phosphate orkaolin), or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin, or olive oil. Powders and granulates may be prepared using theingredients mentioned above under tablets and capsules in a conventionalmanner using, e.g., a mixer, a fluid bed apparatus or a spray dryingequipment.

In various embodiments, the formulations of the disclosure provide avariety of advantages with respect to the prior art. In one aspect, theformulations of the disclosure comprising antigen-specific IgY and acarrier matrix of bovine colostrum have the advantage of being preparedin a rapid time period of within about 6 weeks, once the antigens ofinterest are identified. This allows easy reproducibility andstandardization of the chicken vaccination protocol. In one specificaspect, different flocks of chickens are vaccinated with a single, mixedantigen preparation each, and then combined for a broad spectrumcomposition for the treatment of a pathogenic infection. In one specificaspect, three flocks of chickens are vaccinated with separate mixedantigen preparations then pooled to prepare a broad-spectrum compositionfor the treatment of undifferentiated diarrhea without knowledge of thecausative microbial pathogens. This method has the advantage that themix of antigen-specific antibodies in the composition can be tailoredfor a particular outbreak, region, or season, if desired. Finally, inembodiments, the specific binding molecule need not be separated fromthe whole dried egg for rapid preparation and long term storage.

In another aspect, the compositions of the disclosure are effective fororal administration in the treatment of a pathogenic infection innon-neonates. The gastrointestinal tract of the non-neonate is veryacidic and less absorptive than the neonate, as described herein. In theexamples of the disclosure, the compositions were effective for treatingundifferentiated diarrhea in non-neonatal children of 6 months to 5years of age. In another aspect, the compositions of the disclosure areeffective to treat or prevent traveler's diarrhea in adults. The carriermatrix is a protective and reactive matrix for combination with theantigen-specific binding molecules. In another aspect, the compositionsof the disclosure are provided in a powdered, solid form for suspensionimmediately prior to administration. In one aspect, the suspended, orreconstituted, dosage form has the advantage of being very palatable toinfants and children, even when suffering from the symptoms of apathogenic infection. This has the advantage that the full dose iseasily administered and ingested by the subject suffering from thepathogenic infection.

In another aspect, the compositions of the invention can be used foradministering broad-spectrum passive immunity in either treatment, orprophylaxis of pathogenic infection. In one aspect, a low level ofimmunization of chickens can be sufficient to prepare a composition withan effective amount of anti-antigen specific binding molecule to resultin an effective, broad-spectrum formulation when administered with acarrier matrix.

Treatment or Prophylaxis of Pathogenic Infection

The compositions of the disclosure comprise a specific binding proteinembedded within a carrier matrix. The compositions can be administeredin any method suitable to their particular immunogenic or biologicallyor immunologically reactive characteristics, including oral,intravenous, buccal, nasal, mucosal, dermal or other method, within anappropriate carrier matrix. A specific embodiment involves the oraladministration of the composition of the disclosure.

In various embodiments, the composition is administered as aprophylactic or therapeutic composition. In various aspects, thecomposition includes a pharmaceutically acceptable carrier. In variousaspects, the composition does not include a polymer, copolymer,liposome, hydrogel, or fibrin. In various aspects, the composition doesnot include microspheres or microcapsules. In various aspects, thecomposition does not include an immunogen or antigen. The composition ofthe invention can be administered via oral delivery, nasal deliver,ophthalmic delivery, ocular delivery, mucosal delivery, or a combinationthereof.

One embodiment of this invention uses oral administration. It has beendemonstrated in both human and animal systems that oral (ingested)administration of antibodies, immunoglobulins, and other biologicalimmune factors can have measurable effects on the course, severity andduration on diseases of, in, associated with, or influenced by, thegastrointestinal system.

The admixture of broad-spectrum antibodies is embedded in a within acarrier matrix, such as for example colostrums for oral administration.Colostrum serves to provide synergistic protective and efficaciousattributes to the antibody formulation. Any combination of antibodiescan be used in within a carrier, including but not limited to acombination of anti-pathogen, anti-toxin, and anti-adhesin antibodies.

In one aspect, the compositions of the disclosure are used to treatpatients suffering from various pathogenic infections. The compositionsand formulations for oral administration can be administered once,twice, three times, or four times a day for two, three, four, five, six,seven, eight, nine, 10, 11, or 12 consecutive days for the treatment ofa pathogenic infection. In one aspect, the composition is administeredtwice per day for five days for the treatment of a pathogenic infection.In another specific aspect, the composition is administered once per dayfor three consecutive days for the effective treatment ofundifferentiated diarrhea in non-neonatal children, or in the treatmentof traveler's diarrhea in non-neonatal children or adults. In anotheraspect, the composition may be regularly administered for theprophylaxis of a pathogenic infection.

In the case of a composition for the treatment of a pathogenic infectionof a mucosal membrane by topical administration to a mucosal membrane,the composition can be administered two to six times per day for aperiod of three to 12 days.

In a preferred embodiment, the disclosure provides a compositioneffective for treating undifferentiated diarrhea in non-neonate humans.The composition takes advantage of an effective polyclonal antibodyproduction strategy (chicken innoculation, with antibody harvestingthrough eggs) to generate high specificity antibodies targeted toseveral of the causes of diarrhea pathology. In a specific embodiment,the composition comprises specific polyclonal antibodies in a carriermatrix that is commercial bovine colostrum.

In a preferred embodiment, the disclosure provides an economicalcomposition for the effective treatment of undifferentiated pediatricdiarrhea. The composition comprises a mixture of polyclonal antibodies,primarily IgY, specific for E. coli, Salmonella spp., rotavirus, gramnegative bacteria, toxins produced by pathogens, and adhesins thatenable pathogen attachment and colonization in the gastrointestinaltract.

In a specific aspect, the composition comprises an equivalent weightamount of dried immune egg product from each of three flocks inoculatedwith different antigens or different mixed antigen preparations isco-packaged with a specific weight amount of commercial driednon-hyperimmune bovine colostrum. In one aspect, 0.5 to 3 g. 0.7 to 2.0g, 1.0 g, 1.3 g, or 1.5 g of dried immune egg product from each flock isadded to a single dose packet. Preferably either 1.0 g or 1.3 g eachimmune egg product is added to a one dose packet. In another aspect, 1to 5 g, 2 g to 4 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g or 5g dried colostrum is added to the same packet.

Prior to use, the contents of the packet, or sachet, are mixed intoapproximately 2 ounces of purified water, or some other oral liquid. Theentire reconstituted formulation is administered orally to the subject.The composition can be administered one to four times per day for two toten days. In a specific embodiment, the composition is administered onceper day for 3 consecutive days. The disclosure provides a method oftreating undifferentiated pediatric diarrhea, by administration of thecomposition of the disclosure once per day for two, three or four days.

In one aspect, the composition of the disclosure is administered as anadjunct therapy to antibiotic treatment. In this aspect, the compositionmay be administered once per day for the first three days of treatment.In another aspect, the composition of the disclosure is administeredwith oral rehydration solution (ORS). In another aspect, the compositionof the disclosure is co-administered with an oral zinc formulation. Inanother aspect, the composition of the disclosure is administered as anadjunct to antibiotic treatment to prevent overgrowth of a particularpathogenic organism that is resistant to the antibiotic. As described indetail in the examples, the composition and method is effective torapidly resolve the symptoms of undifferentiated pediatric diarrhea,resulting in significantly decreased stool volume, stool frequency andduration of diarrhea, as well as significantly improved physicianreported well-being.

In one alternative embodiment, the compositions of the disclosure areused to treat traveler's diarrhea. The onset of TD usually occurs withinthe first week of travel but may occur at any time while traveling, andeven after returning home. The most important determinant of risk is thetraveler's destination. High-risk destinations are the developingcountries of Latin America, Africa, the Middle East, and Asia. Personsat particular high-risk include young adults, immunosuppressed persons,persons with inflammatory-bowel disease or diabetes, and persons takingH-2 blockers or antacids. Most TD cases begin abruptly. The illnessusually results in increased frequency, volume, and weight of stool.Altered stool consistency also is common. Typically, a travelerexperiences four to five loose or watery bowel movements each day. Othercommonly associated symptoms are nausea, vomiting, diarrhea, abdominalcramping, bloating, fever, urgency, and malaise.

Infectious agents are the primary cause of TD. Bacterial enteropathogenscause approximately 80% of TD cases. The most common causative agentisolated in countries surveyed has been enterotoxigenic Escherichia coli(ETEC). ETEC produce watery diarrhea with associated cramps andlow-grade or no fever. Besides ETEC and other bacterial pathogens, avariety of viral and parasitic enteric pathogens also are potentialcausative agents.

In one aspect, the composition of the disclosure is administered to thesubject once per day for three consecutive days as an alternative oradjunct to antibiotic treatment of traveler's diarrhea. Limited fieldstudy evidence suggests improvement in diarrheal symptoms within 24 or48 hours of the first dose. Alternatively, two doses per day of thecomposition of the disclosure are administered on day 1, followed by asingle dose on days 2 and 3. In one aspect, the composition of thedisclosure is administered on an alternate daily or weekly schedule, oron a reduced dosage schedule to for prophylaxis of traveler's diarrhea.

In another alternative embodiment, the compositions of the disclosureare used to as a “prebiotic” for gastrointestinal flora management of asubject, for example, prior to administration of a probiotic. As usedherein, the term “prebiotic” refers to a composition that allowsspecific changes, both in the composition and/or the activity of thegastrointestinal microflora that confers benefits upon the subject'swell-being and health. In one aspect, the composition is useful tomanage gastrointestinal flora so as to reduce or eliminate one or moreundesirable strains of bacteria. In one aspect, the anti-antigenicimmunoglobulin composition is tailored to manage gastrointestinal floraso as to reduce or eliminate one or more undesirable strains ofbacteria. In another aspect, the compositions are used as an adjunct totraditional prebiotics. In a further aspect, the composition of thedisclosure further comprises a soluble fiber. In a further aspect thecomposition is used alone for flora management.

In another aspect, the disclosure provides a method of gastrointestinalflora management in a subject comprising the steps of administering thecomposition of the disclosure to reduce or eliminate one or moreundesirable strains of bacteria, followed by administering a probioticto introduce one or more desirable strains of bacteria. In anotheraspect, the composition of the disclosure is administered as an adjunctto antibiotic treatment to prevent overgrowth of a particular pathogenicorganism that is resistant to the antibiotic.

Example 1. Compositions for the Treatment of Diarrhea

Diarrhea is a symptom of a broad range of causes including bacterial,viral, protozoal and parasitic infections. Bacterial diarrhea is inducedby multiple organisms, including various forms of Escherichia coli,Salmonella, Vibrio cholerae and parahemolyticus, Shigella,Campylobacter, Yersinia and others. Viral pediatric diarrhea is oftencaused by Rotavirus, but also may be caused by several other viruses.

There are known to be multiple causative organisms in diarrhea. Thesecausative organisms can be organized into common clusters that producestructurally related toxins, to which a series of broad-spectrumneutralizing antibodies can be created that, when admixed into aformulation with clinically effective titers, can be used as abroad-spectrum organism-independent therapeutic intervention fortoxin-mediated diarrhea.

Briefly, antibodies specific to causative organisms of diarrhea aregenerated by inoculation of chickens with antigen. Immune eggs arecollected and whole egg is pasteurized and spray dried to obtain apowderized form. Commercial bovine colostrum is mixed in a powderizedform. The two powders are added sequentially to a single dose packet andsealed, and distributed in dried form for an oral formulation. Beforeadministration, the powdered oral formulation is mixed with a smallquantity of water prior to oral consumption.

This treatment confers passive immunity to patients, as demonstrated inthe Examples herein. The nature of the treatment makes the associatedrisk factors comparable to that of eating food from the source where theantibodies were harvested (e.g., risk factors would be similar to thatof eating an egg and a glass of milk). This is an effective treatmentwith less toxicity than the currently available alternative medicines.

Example 1A

Chickens were individually inoculated with purified antigens derivedfrom 5 E. coli strains: four ATCC strains, containing E. coli adherencepili antigens F41, 97P, F19 and K99, and one wild type E. coli strainderived from milk. Each chicken was inoculated with only one antigen.Chickens were inoculated once per week for three weeks. Freund'sadjuvant was employed for the first inoculation, followed by Freund'sincomplete adjuvant for the second and third inoculations. Two shots,left and right breast were used per inoculation. Eggs were housedseparately; eggs were collected, flash pasteurized and spray dried. Eachof the five antibody preparations were mixed in equal parts. The driedegg powder anti E. coli antibody preparation was stored frozen for about2 years.

A second flock of chickens was inoculated with a mixed antigenpreparation containing rotavirus, coronavirus and E. coli antigens. Thesame inoculation, collection and egg processing protocols were employedas above. The dried egg powder anti-scours antibody preparation wasstored frozen for 1.5 years. ELISA was used to characterize the antibodypreparations.

One gram each of the dried anti-E. coli antibody preparation and thedried anti-scours antibody preparation were added with 3 grams or 4grams of commercial dried full-fat bovine colostrum in a single dosepacket.

Example 1B

Three flocks of chickens were individually inoculated with one each ofdifferent mixerd antigen preparations: a first antigen preparationcontaining rotavirus (serotypes G6 and G10), coronavirus,enterotoxigenic E. coli stains with K99 pili adherence factor, andClostridium perfringens type C toxoid with adjuvant); a secondpreparation containing enterotoxigenic strains of E. coli having K99,K88, 987P or F41 adherence factors); and a third mixed antigenpreparation containing various E. coli endotoxin; with adjuvant). Eachof three flocks only received a single mixed antigen preparation. Eggswere collected, cleaned, broken, pasteurized and spray dried or thermaldried to create three dried immune egg products. Dried egg product wasoptionally evaluated by ELISA for specific IgY activity. An equal weightof each of the three dried immune egg products was combined with 3 g or4 g of dried colostrum in a single dose packet. Either 2 g, 3 g, or 4 gof combined weight of dried immune egg product was employed per singledose packet, as described below. In one aspect, the commercial driedcolostrum did not exhibit specific activity toward the antigens of thevaccines.

Example 1C

Immunization of chickens for IgY Production.

The following immunization protocol was adapted from a GallusImmunotech, Inc. protocol and can be utilized for generation of IgYpolyclonal antibodies. A few eggs are optionally collected prior toimmunization to serve as a baseline control. If a mixed antigenpreparation for cattle or hogs is employed, it is diluted at 1:2, 1:4,1:8, or 1:16 prior to administration. On day 0, chickens are injectedwith between 0.02 and 0.5 mg antigen with Freund's complete adjuvant.Injections can be either subcutaneous or intramuscularly into the breasttissue of the hen at multiple sites. The total volume ofantigen/adjuvant mixture can be about 1 mL with adjuvant from one-halfto two-thirds of the volume. Immunizations are repeated, typically, ondays 14, 21 and 28, using Freund's incomplete adjuvant, with about halfthe initial amount of antigen. Typically, specific antibody can bedetected at about day 30 in eggs. For prolonged antibody production,hens are boosted every couple of months. Eggs can be stored in coldstorage prior to processing and/or purification of IgY. In one aspect,eggs can be held in cold storage for up to one month, or up to twomonths, prior to processing or purification. In another aspect, IgY canbe generated in a similar fashion in duck, goose, ostrich, quail, orturkey eggs, with use of appropriate amounts of antigen.

Example 2. Ingested Antibody Treatment for Clostridium difficile

In one embodiment, the invention methods and compositions are used totreat Clostridium difficile (C. difficile), a bacterium that isnaturally present in most people. The population levels of C. difficileare kept under control by the other natural flora of the bowel. Patientsoften develop C. difficile infections when antibiotics administered foranother medical condition deplete the natural flora of the bowel,allowing C. difficile populations to multiply unchecked. While manystrains of C. difficile can be treated by specialized antibiotics anincreasingly large number of C. difficile strains are resistant toantibiotic treatment. This leads to a lengthy and difficult recovery forpatients, and may even become life-threatening in certain circumstances.A process neutralizing C. difficile populations with an ingestedantibody that confers passive immunity is capable of controlling C.difficile population levels to allow a natural bowel flora balance to berestored.

As is the case in anti-diarrhea formulations caused by rotavirus andgram-negative bacteria, an antibody embedded in a carrier matrixformulated specifically to bind to C. difficile or its toxins is aneffective therapeutic approach. This formulation can be used to eithertreat an ongoing infection, or to prevent such an infection fromoccurring. Therefore, the treatment can be administered alone, orconcurrently with an antibiotic. This treatment not only benefitspatients recovering from a C. difficile episode, but can be administeredto patients at high risk of developing C. difficile as a prophylactic.

The antibodies that neutralize C. difficile are ingested in a carriermatrix (a mixture of proteins and enzymes that are intended to“activate” the antibody in the bowel, as well as provide usefulsecondary immunity, protection or nutrition). In one embodiment, theantibodies are produced by injecting, or inoculating, an animal with anantigen, or a combination of antigens, which may or may not be containedin a mixed antigen preparation, (potentially combined with an adjuvantto elicit a stronger immune response).

In one aspect, the antigen is obtained from, or derived from, a C.difficile antigen or toxin. In another aspect, the combination ofantigens contains one of more antigens or toxins derived from C.difficile, and one or more additional viral antigens. In another aspect,the combination of antigens contains one or more antigens or toxinsderived from C. difficile, and one or more additional bacterial antigensor toxins. In another aspect, the combination of antigens contains oneor more antigens derived from C. difficile, and one or more additionalprotozoal antigens. In another aspect, the combination of antigenscontains one or more antigens derived from C. difficile, and one or moreadditional fungal antigens.

The antibodies are then either obtained from, isolated from, or derivedfrom, an animal product, such as milk, eggs, or colostrum from theanimal or harvested directly from, the animal, e.g. serum, plasma. In aparticular aspect, hens are inoculated with the antigen, combination ofantigens, or vaccine, and the antibodies are obtained from whole eggs,or egg yolks, or derived from, or purified from whole eggs or egg yolksof the inoculated chickens. In another aspect, the antibodies arepolyclonal antibodies.

This composition is intended to help treat C. difficile infections, orbe a prophylactic against C. difficile infection. For example, thesubstance is comprised of antibodies targeted specifically to C.difficile, embedded within a carrier matrix (for example, colostrum).After harvesting, the antibodies may be powderized. The carrier matrixmay also be powderized. The two powders may then be mixed thoroughly, oradded separately to a single dose packet, or vial, and distributed indried form. In a preferred method of administration, the substance willbe administered orally, by ingestion. To consume, the powdered substancewill be mixed with a small quantity of a liquid, such as water, milk,juice, or electrolyte solution, immediately prior to consumption, andwill be taken as directed by a physician. Other methods of delivery arealso contemplated.

Current treatment for C. difficile infection focuses on antibiotictherapy. However, in cases where strong antibiotics were the cause ofinfection, and in cases where resistance to antibiotics has beendeveloped, few alternative treatments are currently available. Thepresent embodiment seeks to neutralize C. difficile by utilizing naturalimmune mechanisms, rather than toxic antibiotics. It has the advantageof allowing the growth of naturally occurring flora in the bowel whilereducing C. difficile population levels.

The combination of antibodies embedded within a carrier matrix toenhance the effectiveness of the antibodies is not currently used by anyC. difficile disease treatment. The invention methods confer passiveimmunity to patients. The nature of the treatment makes the associatedrisk factors comparable to that of eating food from the source where theantibodies were harvested (e.g., risk factors would be similar to thatof eating an egg and a glass of milk). This is an effective treatmentwith less toxicity than the currently available alternative medicines.

In one aspect, selected antibodies are obtained, purified and isolatedand prepared in a powderized form. In another aspect, the selectedantibodies are not purified, or isolated, but processed as a wholeproduct. For example, the contents of the whole egg obtained from theinoculated chicken is processed, e.g. pasteurized, and prepared in apowderized form, without additional purification steps. An activatingenzyme/protein mixture (for example, including colostrum) is alsoprepared in a powderized form. The two powders are mixed thoroughly anddistributed in dried form for an oral formulation. Beforeadministration, the powdered oral formulation is mixed with a smallquantity of water prior to consumption.

This treatment confers passive immunity to patients. The nature of thetreatment makes the associated risk factors comparable to that of eatingfood from the source where the antibodies were harvested (e.g., riskfactors would be similar to that of eating an egg and a glass of milk).This is an effective treatment with less toxicity than the currentlyavailable alternative medicines.

Example 3. Ingested Antibody Treatment for Helicobacter avlori

Helicobacter pylori (H. pylori) is a gram-negative bacterium which caninhabit areas of the stomach. It is generally believed that H. pylori isassociated duodenal and gastric ulcers and possibly stomach cancer. H.pylori can escape the acidic environment of the stomach lumen byburrowing into the mucus layer of the epithelial cell surface which hasa more neutral pH environment. H. pylori can produce adhesins forbinding to membrane associated lipids or carbohydrates of epithelialcells. Colonization of H. pylori inside areas of the stomach can resultsin chronic gastritis, a long-lasting inflammation of the stomach. Amajor cause of peptic ulcer is H. pylori infection.

Selected antibodies against Helicobacter pylori are obtained andprepared in a powderized form. An activating enzyme/protein mixture (forexample, including colostrum) is also prepared in a powderized form. Thetwo powders are mixed thoroughly, or added separately to single dosepackets or vials, and distributed in dried form for an oral formulation.Before administration, the powdered oral formulation is mixed with asmall quantity of water prior to consumption.

This treatment confers passive immunity to patients. The nature of thetreatment makes the associated risk factors comparable to that of eatingfood from the source where the antibodies were harvested (e.g., riskfactors would be similar to that of eating an egg and a glass of milk).This is an effective treatment with less toxicity than the currentlyavailable alternative medicines.

Example 4. Clinical Studies—Efficacy in Undifferentiated Diarrhea

Effective broad-spectrum treatment of diarrhea is a significantchallenge due to the wide range of causative organisms, the limitedavailability of diagnostic testing for directing treatment regimes.Current standard intervention for cases of severe diarrhea includesubiquitous administration of antibiotics and oral rehydration salts(ORS). However, this approach has shown limited effectiveness, and haspromoted the development of antibiotic resistant bacteria strains.

Example 4A

Field study (trial)s 1 and 2.

Clinical studies were performed to evaluate the tolerability andefficacy of the formulation of Example 1A in treating, or acceleratingthe resolution of undifferentiated diarrhea. A first open,single-center, non-comparative study enrolled a total of 63 pediatricpatients with pediatric diarrhea of both genders between six months andfive years of age. The study compared clinical outcomes of Test Group A,“Trial 1”, receiving the oral formulation of Example 1A, administeredwith antibiotic and ORS, to a Control Group B, receiving only antibioticand ORS. A second Test Group AA, “Trial 2”, enrolled 33 patients in afollow up study to test the formulation of Example 1A under differentseasonal conditions.

All participating pediatric patients presented a “serious” or “severe”diarrhea profile (level 4 or 5) on a 5 point scale (see Table 1), asassessed by attending physician. No diagnostic differentiation was madeas to causative agent or etiology of the pediatric diarrhea. Patientswith rice water stool of bloody stool were excluded. Additionally,patients with known allergies to milk, chicken, or egg products wereexcluded.

TABLE 1 The 5-point scale Level 1 Level 5 Stool Frequency 1-2 per day 10or more per day Stool Consistency 1 = normal 5 = fully liquid PhysicianAssessed Well-being 1 = normal 5 = severe (typically inpatient)

Enrolled children (n=63) were divided into two groups, an experimentalgroup Study 1, “Group A” (34 enrolled children; 29 completing trial),negative control “Group B”, (29 enrolled children; 28 completing trial),and Study 2 “Group AA” (31 enrolled). A second control group “Group BB”receiving antibiotic and ORS was used as a negative control concurrentlywith the Group AA, however, the results are omitted from the figures.

Each test group received 2 g combined egg powder and 4 g colostrum,mixed in water, administered orally once per day for three consecutivedays. Each group was observed and the data are collected for five days.Group A received the composition from Example 1 in addition to astandard regiment of antibiotics and oral rehydration supplements (ORS),as determined by the attending pediatrician. Group B is treated with astandard regimen of antibiotics and ORS. A six month window of timebetween Study 1 and Study 2 was allowed elapse in order to testseasonality. Both trials were conducted in the same study center. Ineach group, antibiotic and ORS prescriptions were determined on acase-by-case basis by the attending pediatrician (Table 2).

TABLE 2 Study Groups with Numbers of Cases Completed Therapy Groupadministered Completed Treatment Period Observation A Composition 29Composition 5 days from Example 1 + from Example antibiotic + ORS 1:days 1-4 Antibiotic + ORS: days 1-6 AA Composition 31 Composition 5 daysfrom Example 1 + from Example Antibiotic + ORS 1: days 1-3 Antibiotic +ORS: days 1-6 B Antibiotic + ORS 28 Antibiotic + 5 days ORS: days 1-5

The composition from Example 1A was packaged in 5 gram powder singledose sachets. The composition was administered orally, with one packetre-suspended in approximately 2 ounces of drinking water. Patients wererequired to drink the entire suspension in one setting, immediatelyafter re-suspension was complete, and this protocol was followed in allcases.

Parameters covered in this example, as measured for each patient,included stool frequency, stool consistency, and physician assessedwell-being. Stool frequency is the guardian or hospital reported numberof diarrheal bowel movements per 24 hour period. Stool consistency is a1-5 scale of consistency with 1 indicating normal and 5 indicatingliquid. Physician assessed well-being is a 1-5 scale of overallcondition with 1 indicating normal parameters for a healthy child and 5indicating a severely ill child.

Physicians participating in the trial were asked to provide theirexperience of the typically patient progression, as measured by thethree parameters described, over the course of six days. The reportedvalues were aggregated into a single expected patient progressionbaseline for each parameter. Patients were evaluated both in terms ofimprovement relative to expected outcomes based on doctor experience,and against the concurrent negative controls.

Data analysis was conducted with MS Excel and Matlab. Statisticalsignificance was computed by a Chi-square test with p-value of <0.05considered significant. Results are shown in FIGS. 1 to 3.

Dramatic improvements in patients receiving the composition from Example1A were observed within 24 hours of the initial dose administration.Within 48 hours after initial dose administration patients weregenerally stabilized at normal or near normal levels.

As shown in FIG. 1, average number of diarrheal bowel movements in a 24hour period decreased from 9 to 2 in Group A (Trial 1) after the initialdose of the composition from Example 1. Group AA (Trial 2) exhibited asimilar reduction from 10 to 3. In contrast, average number of episodesin Group B (Negative control) decreased from 11 to 10 in the same timeperiod. The average number of episodes in Groups A and AA (Trials 1 and2) remained constant at 2 from day 3 onward, while Group B diminishedgradually eventually exhibiting 6 episodes per 24 hours by day five. InGroup A, within 24 hours of the treatment with the composition fromExample 1, stool frequency rates returned to near normal levels,2.32±2.48, an over 86% reduction in the duration of gastroentericsymptoms when compared to the control population (P<0.001). Within 48hours stool frequency rates improve to 2.14±2.19. In Group AA, frequencyrates showed similar stabilization rates, improving to 2.56+/−0.68within 24 hours and 2.00+/−0.45 within 48 hours, a marked improvementcompared to control (P<0.001)

As shown in FIG. 2, iinitial stool consistency was liquid in allpatients. Group A and Group AA stool consistency improved to near-normallevels in 24 hours, after the first dose of the composition, after thefirst dose of the composition from Example 1. Control Group Bconsistency improved but was still liquid in 24 hours, with symptoms notfully resolving the entire observation period. Group A obtained normalstool consistency in 48 hours and for the remainder of the study, whileGroup B, by day 3, improved to mostly liquid. Group B eventually reachednear-normal levels of stool consistency by day 6, while Group A stoolconsistency remained normal throughout days 3-6.

Surprisingly, within 24 hours of the treatment with the composition fromExample 1A, stool consistency rates returned to mild levels, 2.05±1.02for Group A and 1.96+/−0.61 for Group AA, an over 86% reduction in theduration of gastroenteric symptoms when compared to the controlpopulation (P<0.001). Within 48 hours, Group A stool consistency droppedfurther to near normal levels 1.41±0.9, and Group AA levels were1.17+/−0.37.

As shown in FIG. 3, all patients enrolled in the study were rated asseverely ill by attending physicians, up to and including seriousdehydration, vomiting and low responsiveness. Patients in Group A andGroup AA improved significantly overnight (P<0.001), after the firstdose of the composition from Example 1, to an average well-beingassessment level of approximately two. At 24 hours, Group B patientsremained severely ill. Group A and Group AA patients, at 48 hours,improved to near normal and continued to improve on day three obtainingnormal condition, while Group B patients improved but remained very ill.Throughout days 4-6, patients in Group A remain fully recovered whilepatients in Group B improved in a linear manner; however they remainedmoderately ill at the end of the study.

Overall physician reported well-being retuned to near healthy levelwithin one day, with Group A dropping from an initial value of 4.46±0.51 to 1.9±0.9, a level considered within normal parameters for thispopulation. Group AA displayed similar results, falling from an initiallevel of 4.3+/−0.46 to 2.03+/−0.49. This collectively represents an 86%reduction in the duration of illness when compared the controlpopulation (p<0.001). Within 48 hours, physician reported well-beingimproved further to 1.26±0.83 in Group A and 1.4+/−0.49 for Group AA.

A check to confirm the normal distribution of trial cases againstexpected prevalence of Rotavirus was made within Group A (Trial 1),independent of the primary trial evaluation. Stool samples werecollected for 26 of the 29 experimental patients in Group A and 24 ofthe 31 patients from Group AA, and were tested at an independentreference lab using an established commercial agglutination assay(Slidex Rota-kit, bioMerieux, France). Seven of the 26 patients sampledin Group A test positive for Rotavirus (27% of the tested population).This pediatric Rotavirus infection prevalence is in line with expectedresults for the season and the degree of severity of diarrhea casesadmitted to the study. Four of the 24 tested positive in Group AA (17%of the tested population). The prevalence of rotavirus fro Group AA wassomewhat lower than expected. Therefore, the composition of Example 1Awas deemed effective as administered to treat undifferentiated diarrhea,including that caused by rotavirus infection.

To further determine the similarity of response to the composition fromExample 1 between the Rotavirus positive group (RV) and thenon-Rotavirus positive group (Non-RV), the Pearson's Product-MomentCorrelation Coefficient was used (represented as “R), the strength ofwhich is represented in the range −1 to 1. Calculation of R used theaverage of the Non-RV and RV group for each time point, with calculationof the R-value from the average value of each group over the 6 days.

The R value of the RV group's association with the Non-RV group for thePhysician Assessed Well-being dataset is 0.99029, showing a very stronglinear dependence and covariance between the two groups. The behaviorsof Non-RV and RV patients are strongly predictive of each other, andshowed very similar responses to the treatment over the six daytreatment and observation period. These results confirm the efficacy ofthe composition from Example 1 in Rotavirus mediated diarrhea cases(Table 3).

TABLE 3 RV/Non-RV Average Values Day RV Non-RV 1 4.71 4.38 2 1.71 1.95 31.33 1.24 4 1.28 1 5 1.14 1 6 1 1

Of the 96 patients enrolled in the studies, 88 completed the full sixday study period. Four patients were withdrawn from Group A, and twofrom Group AA by the physician when it was determined that theirenteritis was co-morbid with, or the result of, other conditions; asshown in Table 4. One patient from Group A was lost to the trial whenhis guardian decided that the patient was well enough to withdraw afterthe second dose of the composition from Example 1. And, one patient waswithdrawn from Group B due to record keeping error (Table 4).

TABLE 4 Patients Withdrawn from Study Patient # Group Reason Withdrawnby A 12 Experimental (A) Measles Study doctor A 19 Experimental (A)Meningitis 1 A26 Experimental (A) Patient deemed well Guardian A28Experimental (A) Measles Study doctor A33 Experimental (A) MeningitisStudy doctor B08 Control (B) Record keeping error Study doctor

These results suggest that the composition from Example 1 may provide asafe and effective treatment for undifferentiated pediatric diarrhea.Reducing the duration and severity of diarrhea will prevent asignificant amount of morbidity and mortality associated with pediatricdiarrhea and may also help prevent diarrhea-associated co-morbiditiesfrom developing in pediatric patients.

After one day of the treatment with the composition from Example 1,pediatricians report substantial improvement in overall well-being in100% of the patients completing the trial. Surprisingly, significantreduction in both the duration and severity of illness provided an 86%reduction in length of diarrhea episode after two days of the treatmentwith the composition from Example 1. Independent Rotavirus testingconfirmed efficacy of the composition from Example 1A in these cases.

The composition from Example 1A was shown to be highly effective in thetreatment of undifferentiated diarrhea, greatly reducing the length andseverity of illness when compared to conventional therapies alone. Thecomposition from Example 1A is well-tolerated with no adverseside-effects reported. The results of this study represent an importantand robust improvement in the treatment of pediatric diarrhea withindemanding field environments. These results provide an opportunity foradditionally investigation of the mechanisms and biochemistry by whichthe composition of the invention protects patients from the most severesymptoms of undifferentiated diarrhea.

Example 4B

Field study (trial) 3.

A third study trial was conducted with 140 treated patients and 30negative control patients enrolled in Trial 3. The daily dose of thecomposition in the treated arm contained either 2 g total of equalportions of dried whole egg from each of three flocks, each inoculatedseparately with one commercial scours or mastitis vaccine; and 4 gramsof dried bovine colostrum (ES204A; MS204A); or 3 grams of equal portionsby weight of dried whole egg from each of three flocks, each inoculatedseparately with one commercial scours or mastitis vaccine and four gramsof dried bovine colostrum (MS304A). In addition, egg was processedeither by spray drying (S) or thermal drying (T). The flocks were housedat two different geographic locations within the United States (M) or(E).

Trial 3 was conducted as described above; patients were treated with thecompositions once per day, for three consecutive days. Average resultsfor Trial 3 compared to Trials 1 and 2 are shown in FIGS. 4-9. A smallarm of Trial 3 with 15 patients was treated with 2 g dried egg and 4 gcolostrum once per day for two days and exhibited significantimprovement at days one and two in each measured parameter. This groupexhibited a slight average relapse effect in symptom scoring inphysician reported well-being on day 4, stool consistency on days 3 and4 (ES204B). However, these values were still significantly improvedcompared to the negative control group.

These results show that a solid formulation for suspension comprisingspecific binding molecules which are antigen-specific IgY antibodies inwhole dried egg and a carrier matrix, which is non-immune dried bovinecolostrum is economical and effective. The matrix of the disclosure,dried bovine colostrum, is easily commercially available and can providehigher levels of various matrix components than milk. This is incontrast to, for example, the prior art teachings of Larrson et al, US2010/0233162. Larsson provides a method for local administration ofisolated chicken yolk immune globulins (IgY) in human breast milk totreat and prevent fungal infections. At the very least, the use of humanbreast milk makes the Larsson composition less economical and difficultto rapidly produce and store. Further, after three days of treatment,the compositions of the present disclosure are shown to significantlydecrease the duration of undifferentiated diarrhea in non-neonatalbabies and children; where the conditions of the gastrointestinal tractare harsher than in the neonate. This is in contrast to Larsson et al.,US 2006/0134101, which provides a method for the use of avian antibodiesfor treatment and prophylaxis of enteric infections in newborn infants.This is also contrast to, Sarker et al., 2001, who reported a clinicaltrial of hyperimmunized chicken egg yolk immunoglobulin in non-neonatechildren with rotavirus diarrhea that showed little or no difference inthe duration of diarrhea. (Sarker et al., 2001, Randomized,placebo-controlled, clinical trial of hyperimmunized chicken egg yolkimmunoglobulin in children with rotavirus diarrhea. J. Pediatr.Gastroenterol Nutr. 32: 19-25).

In addition, the present compositions utilize dried whole egg containingantigen-specific IgY with a protective and reactive carrier matrix suchas bovine colostrum to both (1) protect the antibodies during oraladministration, and (2) to further activate passive immunity asdescribed. This is in contrast to Lee et al., US 2003/0185856, whichprovides a method for the production of egg containing anti-pathogenicbacteria specific IgY and compositions in the form of yogurt or icecream containing the IgY; however, a protective and reactive carriermatrix is not described. Yogurt and ice cream generally do not have ahigh enough concentration of the matrix components present in the matrixderived from colostrum.

Unlike an immunoregulatory response, the effects of administration ofthe composition could generally be observed within 6-12 hours of thefirst administration. The compositions of the disclosure are effectivewithout reliance on the subject's immune response.

Example 5. Clinical Study—Unexpected Efficacy in Typhoid Fever

Evidence for the efficacy of the claimed composition was providedthrough an unplanned and unexpected demonstration of clinical efficacycaused by an unknown prior inoculation.

During one field study in India a small number of children were broughtforward for treatment who had been clinically diagnosed with “TyphoidFever”. Typhoid fever is an infection most commonly caused by a type ofbacteria called Salmonella typhi (S. typhi). Classical symptoms of thisdisease, beyond diarrhea, are caused by its systemic infection phase.The bacteria typically first travel into the intestines, and then intothe bloodstream, where they can migrate to the lymph nodes, gallbladder,liver, spleen, and other parts of the body. These patients displayedclassical symptoms of advanced disease, including high fever, generalill-feeling, and abdominal pain, and significantly, a classicalrash—“rose spots,” which are small red spots on the abdomen and chest.

As is typical for this practice environment, no diagnostic testing wasperformed on these patients beyond gross physical examination. Althoughthese patients did not fit into the inclusion criteria for the fieldstudy they were provided with the composition of Example 1B, at therequest of the attending physicians, on compassionate grounds.

The standard inoculation protocol for chickens with three commerciallyavailable vaccines did not specifically include antigens for Salmonella,so only a limited clinical response was expected. A mild improvement dueto endotoxin neutralization was predicted, with some associated reliefof the major diarrheal symptoms, but no effect on the course of thedisease itself.

Surprisingly, all of the typhoid fever patients receiving thecomposition of Example 1B showed dramatic improvement in diarrheasymptoms within 24 to 48 hours. This improvement appeared to be beyondwhat might be expected for endotoxin neutralization alone. Moresurprising however was the fact that the systemic symptoms of typhoidfever in all cases also disappeared within the following 24 hour period,yielding a time period to normal or near normal status of 48 to 72hours. In typhoid fever symptoms usually improve in 2 to 4 weeks withtreatment.

None of the patients exhibited any rebound or recurrence of diseaseduring the field trial observation period (5 days). It is wellestablished that symptoms may return rapidly if the treatment has notcompletely cured the infection.

Treatment with the composition of Example 1B, once per day for threeconsecutive was sufficient to cause (in conjunction with standard ofcare) a dramatic reduction in symptoms of the disease, both GI andsystemically, within a remarkably short period of time. The timeframe ofresponse could not be explained by natural or “standard of care” effectsalone.

In an attempt to discover the source of this unexpected efficacy, theentire history of the production process for that lot was carefullyreviewed. It was discovered that as part of an ordinary, butdiscretionary, inoculation protocol for commercial laying hens thechickens we used were inoculated with salmonella vaccine.

Although the birds were vaccinated as chicks, the formulation was foundto be highly efficacious against Salmonella typhi. Salmonella was notone of the antigens in the inoculation protocol used for the chickens inthe Example 1 preparations. The unexpected response of these typhoidfever patients to the composition of the disclosure was noted to be verysurprising to the attending physicians during the field trial.

Example 6. Quantitative ELISA for Egg Powder Specific IgY and Total IgY

The antibody activity of total IgY and specific anti-antigen IgY can bedetermined using Enzyme-Linked Immunosorbant Assay (ELISA) by amodification of the method of Liou et al., 2011, J. Anim. Vet. Adv.,10(18):2349-2356, as described below.

Microtiter plates are coated with either 100 uL mixed antigenpreparation (10 ug per well) or coated with 100 uL rabbit anti-chickenIgY antibody (10 ug/mL, Sigma-Aldrich), for control wells. The plate isincubated overnight at 4° C. After washing with PBS-Tween 20 buffer,plates are blocked with 2% BSA and incubated overnight at 4° C. Thewells are then washed with PBS-Tween 20 buffer and once with PBS.Thereafter, diluted dried egg powder stock (10 mg/mL) is seriallydiluted with 1% BSA and added to sample wells at 100 uL per well. Wellsfor standard curve are filled with 100 uL serial dilutions of standardIgY at, e.g., at concentration ranges of, e.g., 0.015-1 ug/mL andincubated overnight at 4° C. After washing with PBS-Tween 20 buffer, 100uL of alkaline phosphatase-conjugated goat anti-chicken IgY is added tothe wells and incubated 2 hours at 37° C. After washing with PBS-Tween20 buffer, 100 uL disodium p-nitrophenyl phosphate as substrate is addedto each well and allowed to react for 10 min at 37° C. The absorbance ismeasured at 405 nm using a plate reader. The absorbance of standardcurves provides a relative measurement of specific anti-antigen IgYconcentration.

For measurement of total IgY, each well of the microtiter plate iscoated with rabbit anti-chicken IgY antibody (10 ug/mL). Afterincubation and washing as above, 100 uL of diluted dried egg powder isadded and assay is performed as above.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A dosage form comprising a composition for administration to anon-neonate human in need thereof, the composition comprising: a) anon-neonate effective amount of at least one antigen specific antibody,or antigen-binding fragment thereof, obtained from a first nonhumananimal; and, b) a carrier matrix comprising colostrum, or at least twocomponents thereof obtained from a second nonhuman animal, wherein theat least two components are selected from the group consisting ofenzymes, lactoferrin, transferrin, nonspecific immunoglobulins,cytokines, white blood cells, complement components, interferons, growthfactors, and fibronectin, wherein the at least one antigen-specificantibody or antigen-binding fragment thereof and the colostrum or the atleast two components of the carrier matrix are obtained from differentnon-human animals.
 2. The dosage form of claim 1, wherein the carriermatrix comprises bovine colostrum.
 3. The composition of claim 1,wherein the at least two components of the carrier matrix are selectedfrom the group consisting of lysozyme, phospholipase, defensins,opsonins, nonspecific immunoglobulins, proline-rich polypeptides (PRPs),components of the complement system, beta-lysin, lactoferrin,lactoperoxidase, transferrin, cytokines, interleukins, chemokines,interferons, TNF-alpha, fibronectin, leukocytes, white blood cells,phagocytes, macrophages, monocytes, neutrophils, polymorphonuclearcells, dendritic cells, mast cells, eosinophils, basophils, naturalkiller (NK) cells, lymphokine activated killer (LAK) cells, elastase,cathepsin G, myeloperoxidase, NADPH oxidase, insulin-like growth factorI, insulin-like growth factor II, transforming growth factor alpha,transforming growth factor beta 1, transforming growth factor beta 2,fibroblast growth factors, epidermal growth factor,granulocyte-macrophage stimulating growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, colony-stimulatingfactor-I, leptin, hepatocyte growth factor, and combinations thereof. 4.The dosage form of claim 1, wherein the antigen is present in or isderived from a bacterial or viral pathogen, a pathogen related toxin, apathogen related adhesion element, undesirable strain, or a combinationthereof.
 5. The dosage form of claim 4, wherein the bacterial or viralpathogen is a human or veterinary, enteric or gastrointestinal, pathogencapable of causing gastroenteritis.
 6. The dosage form of claim 5,wherein the bacterial or viral pathogen is selected from the groupconsisting of: Campylobacter jejuni, Salmonella, Salmonella typhimurium,Salmonella enterica serovar Typhi, Shigella dystenteriae, Plesiomonasshigelloides, Escherichia coli, enteropathogenic E. coli,enterotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E.coli, haemorrhagic E. coli, Clostridium difficile, Yersiniaenterocolitica, Vibrio cholerae O1, Vibrio O139, Non-O1 Vibrios, Vibrioparahaemolyticus, Aeromonas hydrophila, Clostridium perfringens,enterohepatic Helicobacter, Helicobacter pylori, Staphylococcus aureus,Klebsiella, Gardnerella spp., Neisseria gonorrhoeae, Chlamydiaceaetrachomatis, Mycoplasma spp., Campylobacter jejuni, Trichomonasvaginalis, herpes virus type 1, herpes virus type 2, Candida albicans,Candida glabrata, Candida tropicalis, Candida parapsilosis and Candidakrusei, Group A Streptococcus spp., rotavirus, coronavirus, norovirus,calicivirus, enteric adenovirus, cytomegalovirus, astrovirus, S.pneumoniae, H. influenzae, herpes zoster virus, Fusarium spp., andAcanthamoeba spp.
 7. The dosage form of claim 6, wherein the at leastone antigen-specific antibody, or antigen-binding fragment thereofcomprise a mixture of polyclonal antibodies that are specific for one ormore antigens present in the bacterial and/or viral pathogen, pathogenrelated toxin, or pathogen related adhesion element, derived from one,two, three, four, five, six, seven, or eight, or more, differentpathogenic microorganisms.
 8. The dosage form of claim 4, wherein thepathogen related toxin comprises an endotoxin or exotoxin.
 9. The dosageform of claim 4, wherein the pathogen related adhesion element comprisesadhesins, cadherins, cilia, fimbrillae, a viral adhesion structure, or acombination thereof.
 10. (canceled)
 11. The dosage form of claim 1,wherein the at least one antigen specific antibody or antigen-bindingfragment thereof is selected from a mixture of polyclonal antibodies ora monoclonal antibody.
 12. The dosage form of claim 11, wherein the atleast one antibody is an IgG.
 13. The dosage form of claim 11, whereinthe at least one antibody is an IgY.
 14. The dosage form of claim 11,wherein the at least one antibody is a mixture of polyclonal antibodies.15. The dosage form of claim 2, wherein the bovine colostrum isnon-hyperimmune bovine colostrum.
 16. The dosage form of claim 2,wherein the bovine colostrum is full fat bovine colostrum.
 17. Thedosage form of claim 1, wherein the antigen specific antibody orantigen-binding fragment thereof is in a solid form.
 18. The dosage formof claim 17, wherein the antigen specific antibody or antigen-bindingfragment thereof is crystalline.
 19. The dosage form of claim 1, whereinthe carrier matrix is in a solid form.
 20. The dosage form of claim 1further comprising a pharmaceutically acceptable diluent, binder,excipient, lubricant, sweetening agent, flavoring agent, wetting agent,absorbent, and/or retarding agent. 21.-23. (canceled)
 24. A method forthe treatment or prevention of a pathogenic infection or undesirablestrain of microorganisms in a non-neonate human in need thereof; themethod comprising administration of a composition comprising: a) anon-neonate effective amount of at least one antigen specific antibody,or antigen-binding fragment thereof, obtained from a first nonhumananimal; and, b) a carrier matrix comprising colostrum or at least twocomponents thereof obtained from a second nonhuman animal, wherein theat least two components are selected from the group consisting ofenzymes, lactoferrin, transferrin, nonspecific immunoglobulins,cytokines, white blood cells, complement components, interferons, growthfactors, and fibronectin, wherein the at least one antigen specificantibody or antigen-binding fragment thereof and the colostrum or atleast two components of the carrier matrix are obtained from non-humandifferent animals.
 25. The method of claim 24, wherein the pathogenicinfection is selected from the group consisting of undifferentiateddiarrhea, traveler's diarrhea, rotavirus diarrhea, toxin-mediateddiarrhea, cholera, C. difficile infection, dysentery, typhoid fever, andpeptic ulcers.
 26. The method of claim 25, wherein the undifferentiateddiarrhea is pediatric undifferentiated diarrhea.
 27. The method of claim24, wherein the composition is administered in an amount effective forconferring passive immunity to a subject.
 28. The method of claim 24,wherein the treatment or prevention of an undesirable strain ofmicroorganisms is used for gastrointestinal flora management.
 29. Themethod of claim 24, wherein the pathogenic infection is caused by amicroorganism selected from the group consisting of: Campylobacterjejuni, Salmonella, Salmonella typhimurium, Salmonella enterica serovarTyphi, Shigella dystenteriae, Plesiomonas shigelloides, Escherichiacoli, enteropathogenic E. coli, enterotoxigenic E. coli,enteroaggregative E. coli, enteroinvasive E. coli, haemorrhagic E. coli,Clostridium difficile, Yersinia enterocolitica, Vibrio cholerae O1,Vibrio O139, Non-O1 Vibrios, Vibrio parahaemolyticus, Aeromonashydrophila, Clostridium perfringens, enterohepatic Helicobacter,Helicobacter pylori, Staphylococcus aureus, Klebsiella, Gardnerellaspp., Neisseria gonorrhoeae, Chlamydiaceae trachomatis, Mycoplasma spp.,Campylobacter jejuni, Trichomonas vaginalis, herpes virus type 1, herpesvirus type 2, Candida albicans, Candida glabrata, Candida tropicalis,Candida parapsilosis and Candida krusei, Group A Streptococcus spp.,rotavirus, coronavirus, norovirus, calicivirus, enteric adenovirus,cytomegalovirus, astrovirus, S. pneumoniae, H. influenzae, Neisseriagonorrhoeae, herpes zoster virus, Fusarium spp., and Acanthamoeba spp.30. (canceled)
 31. The method of claim 24, wherein the colostrum is fullfat bovine colostrum.
 32. The method of claim 24, wherein the colostrumis non-hyperimmune bovine colostrum.
 33. The method of claim 24, whereinthe at least two components of the carrier matrix are selected from thegroup consisting of lysozyme, phospholipase, defensins, opsonins,nonspecific immunoglobulins, proline-rich polypeptides (PRPs),components of the complement system, beta-lysin, lactoferrin,lactoperoxidase, transferrin, cytokines, interleukins, chemokines,interferons, TNF-alpha, fibronectin, leukocytes, white blood cells,phagocytes, macrophages, monocytes, neutrophils, polymorphonuclearcells, dendritic cells, mast cells, eosinophils, basophils, naturalkiller (NK) cells, lymphokine activated killer (LAK) cells, elastase,cathepsin G, myeloperoxidase, NADPH oxidase, insulin-like growth factorI, insulin-like growth factor II, transforming growth factor alpha,transforming growth factor beta 1, transforming growth factor beta 2,fibroblast growth factors, epidermal growth factor,granulocyte-macrophage stimulating growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, colony-stimulatingfactor-I, leptin, hepatocyte growth factor, and combinations thereof.34. The method of claim 33, wherein the at least two components of thecarrier matrix include a growth factor and an antimicrobial factor. 35.The method of claim 24, wherein the antigen is present in or is derivedfrom a bacterial or viral pathogen, a pathogen related toxin, a pathogenrelated adhesion element, undesirable strain, or a combination thereof.36. The method of claim 35, wherein the bacterial or viral pathogen is ahuman or veterinary, enteric or gastrointestinal, pathogen capable ofcausing gastroenteritis.
 37. The method of claim 36, wherein thebacterial or viral pathogen is selected from the group consisting of:Campylobacter jejuni, Salmonella, Salmonella typhimurium, Salmonellaenterica serovar Typhi, Shigella dystenteriae, Plesiomonas shigelloides,Escherichia coli, enteropathogenic E. coli, enterotoxigenic E. coli,enteroaggregative E. coli, enteroinvasive E. coli, haemorrhagic E. coli,Clostridium difficile, Yersinia enterocolitica, Vibrio cholerae O1,Vibrio O139, Non-O1 Vibrios, Vibrio parahaemolyticus, Aeromonashydrophila, Clostridium perfringens, enterohepatic Helicobacter,Helicobacter pylori, Staphylococcus aureus, Klebsiella, Gardnerellaspp., Neisseria gonorrhoeae, Chlamydiaceae trachomatis, Mycoplasma spp.,Trichomonas vaginalis, herpes virus type 1, herpes virus type 2, Group AStreptococcus spp., rotavirus, coronavirus, norovirus, calicivirus,enteric adenovirus, cytomegalovirus, astrovirus, S. pneumoniae, H.influenzae, herpes zoster virus.
 38. The method of claim 37, wherein thebacterial or viral pathogen is selected from the group consisting of E.coli, rotavirus, and coronavirus.
 39. The method of claim 37, whereinthe at least one antigen-specific antibody, or antigen-binding fragmentthereof comprise a mixture of polyclonal antibodies that are specificfor one or more antigens present in the bacterial and/or viral pathogen,pathogen related toxin, or pathogen related adhesion element, derivedfrom one, two, three, four, five, six, seven, or eight, or more,different pathogenic microorganisms.
 40. The method of claim 39, whereinthe mixture of polyclonal antibodies comprise IgY antibodies specificfor at least enterotoxigenic E. coli spp., E. coli K99 pili adherencefactor, Clostridium perfringens toxoid, Salmonella typhimurium,rotavirus, and coronavirus.
 41. The dosage form of claim 1, wherein thedosage form is in a form selected from the group consisting of powder,tablet, capsule, troche, or liquid.
 42. The dosage form of claim 1,wherein the dosage form is a solid dosage form.
 43. The dosage form ofclaim 1, wherein one dose of the composition comprises from 1 g to 7 gdried immune egg and from 1 g to 7 g dried bovine colostrum.
 44. Thedosage form of claim 1, that is an oral dosage form.